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Full text of "Studies on conceptus hormone production during the period of mammogenesis and lactogenesis in the cow (Bos taurus) and pig (Sus scrofa)"

STUDIES ON CONCEPTUS HORMONE PRODUCTION DURING THE PERIOD 

OF MAMMOGENESIS AND LACTO GENES IS IN THE COW 

(Bos taurus) AND PIG (Sus scrofa) 



By 
RONALD SCOTT KENSINGER 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF 

THE UNIVERSITY OF FLORIDA 
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1982 



ACKNOWLEDGEMENTS 

The author would like to thank the following people for support 
during the course of these studies. The Dairy Science Department was 
appreciated for providing facilities and an assistantship. The Animal 
Science Department facilities also were used extensively and appre- 
ciated. 

The author wants to acknowledge the considerable support which was 
received from his advisor. Dr. Robert J. Collier, over the years. His 
professional approach to science has enhanced greatly the author's 
training. Special tribute also needs to be made to Dr. Fuller W. Bazer, 
who presented the opportunity to perform the pig studies, and was in- 
strumental in designing and performing these. 

The remaining members of the graduate committee. Dr. William W. 
Thatcher, Dr. H. Herbert Head, and Dr. Donald Caton, are acknowledged 
for the use of their facilities, for their steady encouragement, for 
their enthusiasm, and for their help in analyzing and interpreting data. 
Dr. R. Michael Roberts and Dr. James D. Godkin are noted for their help 
and technical expertise in the purification of bovine placental lactogen; 
and Dr. Charles J. Wilcox and his students, Dr. Arvind K. Sharma and Ms. 
Nancy Simerl, for considerable help with computer analyses and interpre- 
tation of data. Technical help was provided by Angelita Marianno, 
Ronald Walley, Gail Knight, and Ed Mansfield. To accomplish the total 
mastectomy study, the tremendous mobilization of the physiology laboratory 



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also was appreciated. This group includes Rod Geisert, Candie Stoner, 
Randy Renegar, Dr. Charles Ducsay, Jeff Moffatt, Warren Clark, Harold 
Fischer, Julio Neves, and Catherine Willis. 

During the course of the bovine placental lactogen study. Chuck 
Wallace was of invaluable help in setting up and validating the radio- 
receptor assay and numerous other activities. During this program, 
numerous valuable discussions occurred with Dr. Robert Eley, Frank 
Bartol, and Dr. Gregory Lewis. The friendship and interaction with 
all faculty and students in the Dairy Science Department and the Repro- 
ductive Biology conference were appreciated greatly. 

Final recognition is paid to the author's family who have con- 
stantly provided the encouragement to pursue this program and to the 
author's wife, Peggy, who is a constant inspiration. 



-Ill- 



TABLE OF CONTENTS 

Page 

ACKNOWLEDGEMENTS ii 

ABSTRACT vi 

CHAPTER 

I INTRODUCTION 1 

II REVIEW OF LITERATURE 7 

A. Overview 7 

B. Mammo genes is 7 

Characterization of Maimnogenesis 7 

Endocrine Control of Mammogenesis 11 

Influence of Conceptuses on Mammogenesis 15 

C. Lactogenesis 17 

Characterization of Lactogenesis 17 

Endocrine Control of Lactogenesis 20 

D. Conceptus Hormone Production 24 

III CHARACTERIZATION OF MAMMOGENESIS AND LACTOGENESIS IN 

THE GILT 29 

A. Nucleic Acid, Metabolic, and Histological Changes 
in Gilt Mammary Tissue During Pregnancy and 
Lactogenesis 29 

Introduction 29 

Materials and Methods 30 

Results and Discussion 33 

B. Ultrastructural Changes in Porcine Mammary Tissue 

During Lactogenesis 62 

Introduction. 62 

Materials and Methods 63 

Results and Discussion 64 

C. Summary 79 

IV INFLUENCE OF CONCEPTUSES ON MAMMARY DEVELOPMENT AND 

LACTOGENESIS IN THE PIG 84 

A. Effect of Conceptus Number on Mammary Development 

in Gilts 84 



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Page 

Introduction 84 

Materials and Methods 86 

Results and Discussion 93 

B. Effect of Prostaglandin F2a on Lactogenesis in 
Pseudopregnant Gilts 132 

Introduction 132 

Materials and Methods 134 

Results and Discussion 135 

C. Summary 148 

V PRODUCTION AND ISOLATION OF BOVINE PLACENTAL LACTOGEN. . . 152 

A. Introduction 152 

B. Materials and Methods 155 

C. Results and Discussion 163 

D. Summary 187 

VI GENERAL SUMMARY 191 

APPENDICES 

I AMONG ANIMAL CORRELATION BETWEEN MEAN HORMONE CONCEN- 
TRATION .... 201 

II AMONG ANIMAL CORRELATIONS BETWEEN MEAN HORMONE CONCEN- 
TRATIONS (DAY 10-90) AND MAMMARY VARIABLES IN GILTS WITH 
0, 4-7, OR 8-11 FETUSES 202 

III AMONG ANIMAL CORRELATIONS BETWEEN MEAN HORMONE CONCEN- 
TRATIONS (DAY 70-90) AND MAMMARY VARIABLES IN GILTS WITH 
0, 4-7, OR 8-11 FETUSES 203 

IV WITHIN-GROUP CORRELATIONS BETWEEN MEAN HORMONE CONCEN- 
TRATIONS AND MAMMARY VARIABLES IN GILTS WITH 0, 4-7, 
OR 8-11 FETUSES 205 

V COMPOSITION OF MINIMUM ESSENTIAL MEDIUM UTILIZED FOR 
COTYLEDON EXPLANT CULTURE 207 

VI PROCEDURE FOR PREPARATION OF MICROSOMAL FRACTION FROM 
LACTATING RABBIT MAMMARY GLAND FOR USE IN LACTOGENIC 
RADIORECEPTOR ASSAY 209 

VII PROCEDURES AND VALIDATION FOR LACTOGENIC RADIORECEPTOR 

ASSAY 211 

VIII PROCEDURE FOR CONSTRUCTING SELECTIVITY CURVES FOR DETER- 
MINATION OF MOLECULAR WEIGHTS BY GEL FILTRATION 213 

LITERATURE CITED 214 

BIOGRAPHICAL SKETCH 228 



Abstract of Dissertation Presented to the Graduate Council 
of the University of Florida in Partial Fulfillment of the Requirements 
for the Degree of Doctor of Philosophy 



STUDIES ON CONCEPTUS HORMONE PRODUCTION DURING THE PERIOD 

OF MAMMOGENESIS AND LACTOGENESIS IN THE COW 

( Bos taurus ) AND PIG (Sus scrofa ) 

By 

Ronald Scott Kensinger 

May, 1982 

Chairman; Robert J. Collier 
Major Department: Animal Science 

The initial experiment characterized changes in mammary gland develop- 
ment in gilts during pregnancy. Histology and nucleic acid concentra- 
tions indicated rapid mammary growth from day 75 to 90 of pregnancy. 
Histology, ribonucleic acids, and tissue slice incubation measurements 
showed that stage I lactogenesis occurred between day 90 and 105 of 
pregnancy, and stage II lactogenesis or copious milk secretion occurred 
between day 112 of pregnancy and day four of lactation. Ultrastructure 
indicated mammary epithelial cells were undifferentiated on day 90 of 
pregnancy, began differentiation by day 105, and completed differentia- 
tion by day four of lactation when cells had abundant rough endoplasmic 
reticulum, rounded nucleus, vacuolated Golgi apparatus, and numerous 
apically located lipid droplets and secretory vesicles. Effect of con- 
ceptus number on maternal hormone concentrations and mammary development 
during pregnancy also was examined. Positive associations were detected 
between conceptus nximber and maternal concentrations of estrogen-sulfate, 
estrone, and estradiol, but not prolactin or progesterone. Increases 
in maternal concentrations of estrogens were coincident with previously 
detected rapid mammary gland growth. Estrogens were associated positively 



-vx- 



with each other but not with progesterone or prolactin. Mammary develop- 
ment was reduced markedly in pseudopregnant gilts, but deoxyribonucleic 
acid (DNA) contents did not differ between glands from gilts with four 
to seven conceptuses or eight to eleven conceptuses. Positive among 
animal correlations existed between estrogen concentrations and mammary 
wet weight, and nucleic acids. Positive among animal correlation existed 
between prolactin and mammary wet weight. Qxiadratic relationship existed 
between conceptus number and mammary gland DNA, suggesting that conceptus 
presence was necessary for normal mammary development but that larger 
litters had no additional benefit. Results suggest estrogens of con- 
ceptus origin stimulated mammogenesis during pregnancy. In the final 
experiment bovine placental lactogen (bPL) was produced by culturing 
bovine cotyledons, isolated and purified from culture medium. Lactogenic 
activity was monitored with radioreceptor assay, and purification 
accomplished with gel filtration and ion exchange chromatography. 
Results indicate bPL is a polypeptide hormone of molecular weight 
34,000 by gel filtration or 36,000 by SDS electrophoresis. Isoelectric 
point of protein was 6.2 to 6.4, and migrated as a group of three very 
similar proteins. Evidence suggests partially purified hormone was not 
stable to lyophilization as aggregation or polymerization occurred with 
a resulting loss in biological potency. Lactogenic activity of bPL was 
demonstrated in mammary tissue explant culture bioassay. 

Results suggest estrogen may be the mammogen of conceptus origin 
during pregnancy in the pig. A lactogenic hormone (bPL) was isolated 
from bovine fetal placenta secretion. The role of bPL in mammogenesis 
during pregnancy in cattle remains to be determined. 



-vii- 



CHAPTER I 
INTRODUCTION 

In an evolutionary sense, the process of lactation is a novel ex- 
tension of the reproductive process. Fetal dependence on the dam is 
extended into the postpartum period allowing continued nourishment of 
the offspring. Milk is thus essential to the survival of newborn 
mammals. Man recognized the high quality of milk as a food source and 
incorporated it into his diet some 8500 years ago with the domestica- 
tion of the goat (Campbell and Lasley, 1969). Since that time, milk 
and milk products have become the basis for a large industry substan- 
tially contributing towards meeting the nutrient requirements of man. 
Thus, maximizing efficiency of milk production in domestic animals 
directly contributes to food availability via milk for human consumption 
and indirectly by influencing growth rate of newborn doestic animals 
which will eventually enter man's meat supply. 

In the past quarter century, world-wide milk production increased 
49% compared to a 53% increase in world population (Dunkley and Pelis- 
sier, 1981). A further increase in population will require increased 
production of all food supplies including milk just to maintain the cur- 
rent level of nourishment. Basic research is required to define and 
overcome limiting factors to milk production. 

In an age when efficiency has become increasingly important in 
every aspect of society, the lactating animal is almost without peer 



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-2- 



amons 



g domestic animals in her efficient conversion of feed energy and 
feed protein to high quality edible animal product (Van Horn et al. , 
1972). 

Although cows produce 91% of the world's current milk supply 
(Dunkley and Pelissier, 1981), milk production is important in other 
species, as well. Buffalo, sheep, and goats are important sources of 
milk for human consumption in some countries because of their ability 
to adapt to certain regions (Kosikowski, 1981). In addition, lactation 
is important for efficient reproduction in all domestic species. Thus, 
there is a need for comparative studies of mammary gland biology in 
addition to research on lactation in dairy cattle. 

Mammary gland growth occurs during the embryonic, fetal, prepuberal, 
and postpuberal periods, and during pregnancy and lactation (Anderson, 
1974). However, in all species examined except the guinea pig the 
majority of mammary growth occurred during pregnancy (Tucker, 1969; 
Hacker, 1970; Anderson, 1974). Therefore, it is important to identify 
those factors present during pregnancy which established the number of 
mammary epithelial cells by parturition. 

From endocrine gland extirpation and hormone replacement experi- 
ments, it was determined that hormones from the ovaries (estrogen and 
progesterone) and pituitary (prolactin and /or growth hormone) stimulated 
mammary development during pregnancy (Anderson, 1974). However, also it 
was known that placentae from laboratory animals were capable of par- 
ticipating actively in mammary development (Lyons, 1958). In a broad 
sense, this was not a new concept. In his drawings of the female repro- 
ductive tract (figure I-l) , Leonardo da Vinci clearly indicated the 
presence of blood vessels running from the uterus to the breasts which 



Figure I-l. A reproduction of Leonardo da Vinci's drawing of the female 
genital tract depicting the uterus-to-breast blood vessels 
(Ramsey, 1977). 



-4- 



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t. ^r- t-ii::^,-^:^'-^ 



-5- 



were thought to carry menstrual blood to the mammary glands during 
pregnancy for conversion to milk. The anatomists of da Vinci's time 
hypothesized a direct humoral link between the uterus and mammary glands. 
While this blood vessel did not really exist, it was known that placen- 
tal lactogens existed in several species of animals (Talamantes et al, , 1980), 
and that conceptuses of several species synthesized steroid hormones 
(Perry et al. , 1973; Eley, 1980; Simpson and MacDonald, 1981). There- 
fore, mechanisms were available for conceptuses to influence mammary 
development in these species. 

The early bovine conceptus can synthesize estrogens (Eley, 1980), 
although in small quantities, and is reported to have a placental lacto- 
gen (Bolander and Fellows, 1976). However, large discrepancies exist 
between the physical and biological characteristics of the bovine pla- 
cental lactogen preparations, and reports of concentrations of the 
hormone in pregnant cows have ranged from less than 5 ng/ml (Schellen- 
berg and Friesen, 1981) to greater than 1000 ng/ml (Bolander and 
Fellows, 1976). 

The porcine conceptus also produces estrogens (Perry et al. , 1973), 
but there is no evidence for porcine placental lactogen synthesis (Talaman- 
tes et al., 1980). This species difference was exploited during 
studies to contrast the effect of the gravid uterus and its concents on 
mammary gland development in two species; one with a placental lactogen 
(cow) , and one without it (pig) . Several studies have reported changes 
in histology, cytology, nucleic acids, enzyme activities, and metabolic 
rates of bovine mammary tissue during mammogenesis and lactogenesis 
(Feldman, 1961; Baldwin, 1966; Mellenberger et al. , 1973). However, 
few such studies have been done in the pig. Therefore, the first 



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-6- 



objective of the current study was to characterize mammogenesis and 
lactogenesis in the pig by describing changes in nucleic acids, histology, 
cytology, and in vitro rates of metabolic activity from gilts during 
gestation and early lactation. The second objective was to examine the 
effect of different numbers of conceptuses on hormone concentrations 
during pregnancy or pseudopregnancy and mammary development in gilts. 
The third objective of the investigation was to produce and isolate 
bovine placental lactogen. Therefore, the general thesis of these 
studies was to examine the effect of conceptuses on mammary development 
during pregnancy in the pig and the cow. 



CHAPTER II 
REVIEW OF LITERATUVE 

A. Overview 

As stated earlier, mammary gland function is related to the stage 
of reproductive function in the female mammal. This is evident when 
considering the fact that male mammals do not lactate. Furthermore, 
the majority of mammary growth in the female occurs only during preg- 
nancy and lactation. Thus, there is an obvious relationship between 
the presence of a conceptus and mammary growth, and close coupling of 
the onset of lactation with the process of parturition. The objective 
of these studies was to examine maternal hormone concentrations and 
conceptus hormone production during the process of mammogenesis and 
lactogenesis in the cow ( Bos taurus ) and pig ( Sus scrofa ) with the in- 
tent of identifying possible mammogenic or lactogenic hormone (s). There- 
fore, a discussion of mammogenesis, lactogenesis, and hormone production 
by the conceptus will follow. 

B. Mammogenesis 
Characterization of Mammogenesis 

The various methods used to characterize mammogenesis have included 
macroscopic methods (such as palpation, wet weights, and whole mount 
preparations), microscopic methods, and biochemical methods (as in DNA 



-7- 



measurements) (Munford, 1964). Turner (1952) has outlined the develop- 
ment of the mammary gland in the cow. During the first month of embryonic 
life the mammary gland consists of a single layer of ectodermally derived 
cuboidal cells which differentiate from the underlying mesenchyme. The 
mammary bud forms during the second month of life, and rapid prolifera- 
tion of cells in the Malpighian layer forms the primary sprout during 
the third month of fetal life (Turner, 1952). The gland cistern is 
formed by the separation of cells in the center of the primary sprout 
and enlarges as the lining layers of epithelial cells proliferate. 
Secondary sprouts of cells are given off from the terminal end of the 
primary sprout at 13 weeks of fetal life, and branch into the underlying 
mesenchyme (Turner, 1952). Secondary sprouts eventually give rise to 
tertiary sprouts which, in turn, canalize to form the duct system of 
the udder. However, little growth beyond the secondary sprout stage 
occurs in the fetus. Similar descriptions of mammary duct formation 
have been provided for mice (Raynaud, 1961) and pigs (Turner, 1952) with 
the exception that the teat of the pig contains two ducts. 

Mammary gland growth from birth to puberty involves large increases 
in the amounts of adipose and connective tissue. However, increases in 
epithelial cell mass also occurred, and in most species a period of 
isometric growth (growth at the same rate as the body) was followed by 
a period of allometric growth (greater rate than body growth) of the 
gland up to puberty (Schmidt, 1971). Studies from laboratory animals 
(Mayer and Klein, 1961) and heifers (Sinha and Tucker, 1969) indicated 
that in each estrous cycle mammary gland ducts proliferate in response 
to estrogen while the lobulo-alveolar tissue grows in response to pro- 
gesterone. Ducts and alveoli each undergo regression during each cycle 
but a net growth occurred over time in all species. 



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-9- 



In all mammals, with the possible exception of the guinea pig, the 
majority of mammary gland growth occurred during pregnancy (Hacker, 
1970; Anderson, 1974). Presence of estrogen and progesterone during 
this period stimulated formation of secretory units (alveoli) on the 
ducts which comprised a lobule (Lyons, 1958). Ductal growth along con- 
nective tissue bands occurred in mammary glands of all species examined 
in early pregnancy (Schmidt, 1971). In midpregnancy, alveoli or end-bud 
development increased to replace the adipose tissue in the gland, with 
further development of ducts, alveoli, vascular, and lymphatic systems 
during late gestation (Schmidt, 1971). Progressive increases in mammary ; 

DNA content have been observed during pregnancy in several species (Tucker, 

I 
1969; Anderson, 1974; Anderson et al., 1981). Increases in mammary gland 

DNA during lactation range from 2% in sheep to 40% in the mouse (Ander- 
son, 1975b). ; 

t 
Mammary gland growth during pregnancy in the cow has been described j 

by Hammond (1927), Kwong (1940), and Swanson and Poffenbarger (1979). 
Hammond showed through a series of stained saggital sections of udder 
halves that alveolar growth was greater than ductule growth during preg- 
nancy. The proportion of parenchjnna to fat was small during the first \ 

I 
I 

three months of gestation, but greater than that observed in virgin ; 

heifers (Hammond, 1927). Progressive increases in parenchyma were noted 

in the fourth, fifth, and sixth months of gestation and the gland at 

this stage of pregnancy was similar to that reported by Hammond (1927) 

in the last trimester. Unf ortimately , animals utilized by Hammond were 

not of dairy breeds and were not standardized by age. Kwong (1940) i 

studied histological appearance of mammary tissue in heifers and cows ' 

from one to nine months gestation. The first three months of gestation 



-10- 



comprised the period of duct growth, and the fourth to seventh month 
was characterized by lobulo-alveolar development (Kwong, 1940). Secre- 
tion was first observed at approximately seven months of gestation, and 
increased until term. The appearance of secretion noted by Kwong (1940) 
occurred at the stage of pregnancy when Hammond (1927) noted an increase 
in udder weight of greater than 50% per month. 

Swanson and Poffenbarger (1979) were the first to report data on 
month to month increases in total mammary gland DNA in pregnant heifers. 
While heifers in this study varied considerably in age at slaughter (18 
to 36 months) , data were adjusted for differences in body weight before 
generating growth equations (Swanson and Poffenbarger, 1979). Results 
suggested that mammary gland growth was a continuous exponential process 

throughout gestation described by the equation for organ growth, Y = 

kt 
Ae , in which k is the rate constant for growth by months (t) . Swanson 

and Poffenbarger (1979) indicated growth did not decline in the last 
trimester as indicated by Hammond (1927). 

Studies on mammary development in the pig have been reported by 
Turner (1952) and Hacker (1970). On day 30 of pregnancy there were in- 
creases in lateral buds and sprouts of the duct system (Turner, 1952). 
On day 45 of pregnancy the predominant feature of mammary histology was 
increased development of alveoli within the lobules, and by day 60 
lobulo-alveolar growth was almost complete. On day 75 of pregnancy, 
Turner noted that mammary growth was complete and that secretion was 
beginning to accumulate within the lumina of ducts and some alveoli. 
By day 90 of pregnancy, secretions within the gland had increased con- 
siderably and the alveolar lumina began to expand (Turner, 1952). By 
day 105, the alveoli were very large owing to the accumulation of 



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colostrum, and by the day of parturition lactation had begun as indicated 
by lobules and alveoli which were completely filled (Turner, 1952). 

Hacker (1970) reported that total mammary gland DNA was 1.7 g in 
gilts during the second estrus, and only 1.4 g on days 25 and 50 of 
gestation. However, mammary DNA had increased to 8.1 g/gilt by day 100 
of pregnancy (Hacker, 1970), apparently reflecting the lobulo-alveolar 
development described by Turner (1952) on days 45, 60, and 75. In a 
second study Hacker (1970) compared mammary DNA in gilts and sows on day 
110 of pregnancy (day 110), the day of parturition (day 0), and the 
second day of lactation (day 2). Total DNA averaged 18.7, 24.3, and 24.2 
g in gilts on day 110, 0, and 2, respectively, compared to 13.0, 22.4, 
and 27.9 g in sows (Hacker, 1970). Neither parity nor day significantly 
affected DNA content (Hacker, 1970), suggesting that mammary gland DNA 
was maximal on day 110 of pregnancy. It also is possible that differ- 
ences between days 110, 0, and 2 would have been significant if more than 
two animals per group were used. 

Endocrine Control of Mammogenesis 

Numerous investigations have indicated that estrogen and progester- 
one are Involved initimately in mammogenesis (Lyons, 1958) and the develop- 
ment of the characteristic lobulo-alveolar structure necessary for lacto- 
genesis. Allometric growth of mouse mammary glands was observed just 
prior to puberty and could be abolished by ovariectomy, and restored 
with estrogen injections (Forsyth and Hayden, 1977). In hypophysectomized, 
adrenalectomized, ovariectomized rats with an undeveloped mammary duct 
system, estrogen + growth hormone + adrenal steroid were required to 
develop the gland to a comparable degree observed at puberty. Progesterone 



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-12- 



and prolactin were required to elicit lobulo-alveolar development com- 
parable to that observed in the second half of pregnancy (Forsyth and 
Hayden, 1977). Because of interpretation problems in intact animals, 
much of what is known about the endocrine requirement for mammogenesis 
was determined from studies in endocrinectomized animals or in vitro . 

Mammary tissue from prepuberal mice will grow in cultures containing 
insulin, prolactin, and a corticoid only if animals are first given 
priming injections of estrogen and progesterone (Ichinose and Nandi, 
1966) . Tissue from three to four week old (prepuberal) rats exhibited 
extensive lobulo-alveolar growth when cultured in medium containing 
insulin + estrogen + progesterone + prolactin + aldosterone (Dilley and 
Nandi, 1968). Therefore, the minimum requirement for lobulo-alveolar 
growth in vitro was insulin + prolactin + aldosterone, and only after 
prior exposure to ovarian steroids (Wood et al. , 1975) . 

Stoudemire et al. (1975) demonstrated through the use of tritiated- 
thymidine incorporation into mammary tissues in culture that prolactin 

was required for stimulation of mammogenesis by ovarian steroids. They 

3 

also determined estrogen plus prolactin favored H incorporation in 

ductal epithelium, while progesterone plus prolactin enhanced incorpora- 

3 
tion of H into alveolar epithelium. 

Pituitary protein hormones are required for maximal lobulo-alveolar 
development, and can induce some growth in ovariectomized, adrenalec- 
tomized, hypophysectomized animals if given in sufficient doses (Tal- 
walker and Meites, 1961). 

The positive effect of prolactin on lobulo-alveolar development in 
numerous species has been related (Lyons, 1958; Anderson, 1974; Forsyth 
and Hayden, 1977; Delouis et al. , 1980). It appears that growth hormone 



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alone, or in combination with ovarian steroids, also has the ability to 
stimulate mammogenesis in ovariectomized rats (Harness and Anderson, 
1977a). Earlier studies had indicated this, but contamination of the 
preparations with prolactin was a problem. Certain strains of mice also 
demonstrated a mammogenic response to ox growth hormone (Nandi and Bern, 
1960). 

The role of insulin in mammogenesis originally was believed to be 
critical to stimulate epithelial cell division (Lockwood et al. , 1967); 
however, the demonstration that mitosis was not sustained in cultures 
maintained for several days (Wood et al., 1975) indicated that the role 
of insulin was a permissive one. 

The fact that both insulin and adrenal steroid were required for 
mammary tissue survival in vitro had been established (Elias, 1959). 
Adrenal steroids also have been reported to enhance the formation of 
lobules (Topper and Freeman, 1980), but Nandi (1958) has shown that 
adrenalectomy caused relatively little loss of alveoli in mature virgin 
mice. 

While thyroid hormones have not received much attention with respect 
to their mammogenic properties, there are data which suggest that thyroid 
hormones may stimulate lobulo-alveolar development (Mixner and Turner, 
1942; Vonderhaar and Greco, 1979). It is not known if thyroid hormones 
stimulate mammogenesis during pregnancy. 

Relaxin has been reported to enhance lobulo-alveolar development in 
intact or ovariectomized animals injected with estrogen or progesterone 
(Wada and Turner, 1959a, b). Smith (1954) reported that thyroidectomy 
abolished the growth promoting effect of relaxin which suggested that 
the effect of relaxin was mediated via the thyroid. Recently, Harkness 



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and Anderson (1977a, b) reported that relaxin has limited mammotropic 
activity alone, but that it synergizes with prolactin, growth hormone, 
and ovarian steroids to increase mammary development in ovariectomized, 
hypophysectomized rats. It would be revealing to examine the effect of 
purified relaxin on mammary tissue in culture. 

Hypophysectomy during pregnancy indicated that placentae of several 
species are capable of producing a mammogenic hormone (Anderson, 1974) . 
Indeed, over forty years ago Newton and Beck (1939) demonstrated mammo- 
genic activity in the mouse placenta. Since that time, evidence for the 
existence of placental lactogen has been gathered in numerous species (Talaman- 
tes et al. , 1980), and data suggest that it (at least human placental 
lactogen) can substitute for prolactin to induce both epithelial growth 
and differentiation (Topper and Freeman, 1980) . Further discussion of 
placental lactogen will follow later in this chapter. 

A discussion of the endocrine control of lactogenesis would not be 
complete without mention of hormone receptors, since these factors 
ultimately may regulate tissue responsiveness (Forsyth and Hayden, 1977) . 
The estrogen receptor of the mammary gland is induced by prolactin and 
inhibited by progesterone in laboratory species (Forsyth and Hayden, 
1977; Tucker, 1981). Binding of estrogen by the mammary gland increased 
at lactogenesis. The progesterone receptor was high in mammary tissue 
of the virgin animal, decreased during pregnancy, and was low or non- 
detectable during lactation (Forsyth and Hayden, 1977). There has been 
a report that progesterone binding is decreased by prolactin (Topper 
and Freeman, 1980) . The prolactin receptor concentration is greater in 
rabbits than in species which have a placental lactogen, suggesting that 
receptors are masked in the latter group (Forsyth and Hayden, 1977). It 



-15- 

is known that mammary prolactin receptors increase at midpregnancy in 
rabbits (McNeilly and Friesen, 1977), but not until parturition in rats 
(Hayden at al., 1979). It also has been determined that prolactin it- 
self, and estrogen, induce the prolactin receptor, while progesterone 
antagonizes this effect. The significance of greater numbers of estrogen 
receptors during lactation is not known. It is possible that the peri- 
parturient rise in prolactin induces the estrogen receptor during lacto- 
genesis, but for what reason is unclear. That mammary tissue progesterone 
binding decreases from conception through pregnancy is a paradox. One 
might presume from the well-established effect of progesterone on alveolar 
development, that progesterone binding would be highest during late 
gestation, when mammary alveoli were most abundant. Such is not the 
case. In contrast, the binding of prolactin to the mammary glands of 
rats and rabbits tends to parallel its lactogenic effects. Clearly, 
considerable work needs to be done in the area of hormone receptor 
quantification before their physiological significance can be realized. 

Influence of Conceptuses on Mammogenesis 

Lyons (1958) emphasized the importance of placental hormones to 
stimulate mammogenesis during pregnancy. While the pituitary polypep- 
tides prolactin and growth hoirmone were very effective at promoting 
mammogenesis experimentally, the majority of mammary gland growth during 
pregnancy occurred when concentrations of these hormones were low. Re- 
ports by Bo lander et al. (1976) and Hayden et al. (1979) that lactogenic 
activity in the circulation was positively correlated with postpartum 
milk production generated much interest. Placental lactogen concentra- 
tions in goats (Hayden et al. , 1979) and mice (Markoff and Talamantes, 



-16- 

1981) were related directly to the number of young in utero . This could 
provide a mechanism by which the conceptuses could ensure adequate 
nutrition postpartum. It is known that number of offspring influences 
milk production in sheep (Butler et al., 1981), and birth weights of 
dairy calves were positively associated with subsequent milk yields of 
the dam (Erb et al. , 1980; Thatcher et al., 1980). In contrast, no 
positive effect of twinning on subsequent lactation was observed by 
Chapin and Van Vleck (1980), possibly because of reduced gestation length 
in cattle with twins. 

The quantitative contribution of placentae to mammary gland growth 
in rats and mice has been investigated by several laboratories using 
mammary gland weight or DNA as indices of growth. Nagasawa and Yanai 
(1971) surgically adjusted the number of mouse conceptuses (from one to 
12) on day eight of pregnancy and quantified total mammary DNA and RNA 
on day 20. Results showed a significant positive correlation between 
the number and weight of placentae and mammary development. Studies of 
rats by Desjardins et al. (1968) indicated that conceptuses (and, in 
particular, placentae) were the major stimulus to mammary development 
in the second half of gestation. Further evidence that pituitary pro- 
lactin was not the limiting stimulus to mammogenesis (in the rat) was 
provided by Anderson (1975a). Mammary development in rats on day 20 of 
pregnancy was the same in animals which were hypophysectomized on days 
11, 12, or 15 of pregnancy as it was in sham-operated controls. In addi- 
tion, it was shown that a minimum of three fetal placental units were 
necessary for normal mammogenesis (Anderson, 1975a) in the absence of 
the pituitary. No designed experiments to examine conceptus effects on 
mammary development in the domestic pig have been conducted. 



-17- 

C, Lactogenesis 
Characterization of Lactogenesls 

Lactogenesis is defined as the initiation of milk secretion. How- 
ever, this definition probably is not adequate for the student of mammary 
gland biology. The timing of the event is dependent upon the parameter 
used to establish its occurrence (Cowie et al. , 1980). Because secre- 
tory activity first can be detected at various stages from midpregnancy 
onwards, Fleet et al. (1975) distinguished between lactogenesis stage I, 
the gradual appearance of colostrum in the gland, and lactogenesis stage 
II, the copious milk secretion near parturition. Lactogenesis is a time 
when the mammary epithelial cells undergo remarkable changes in cellu- 
lar structure and biochemical composition. Ultrastructural character- 
ization of undifferentiated and differentiated mammary epithelial cells 
has been done by Heald (1974) and by Hollmann (1974), and an excellent 
review on comparative mammary fine structure has been published (Wooding, 
1977). In fact, there are few species differences in mammary ultra- 
structure, and the transformations in ultrastructure during lactogenesis 
are quite consistent among species (Wooding, 1977). The ultrastructure 
of the prelactating mammary epithelial cell was characterized by an 
irregularly shaped nucleus, a minimal amount of endoplasmic reticulum, 
small and inconspicuous Golgi apparatus, few microvilli on the apical 
cell membrane, few mitochondria throughout the cytoplasm, and commonly 
one or more milk fat droplets in the apical cytoplasm (Heald, 1974; 
Hollmann, 1974). In contrast, the epithelial cell at the time of par- 
turition had undergone extensive differentiation and was characterized 
as having a round, smooth-membraned nucleus, abundant rough endoplasmic 



reticulum, Golgi vacuoles containing casein micelles, conspicuous micro- 
villi on the apical plasma membrane, an increased number of mitochondria 
and cytoplasmic fat droplets, and a distinct cellular polarity resulting 
from the position of the nucleus, rough endoplasmic reticulum, and Golgi 
apparatus (Heald, 1974; Saake and Heald, 1974). 

Concomitant with ultrastructural changes which occur in the mammary 
epithelial cell during lactogenesis, the activities of key enzymes in- 
volved in milk synthesis increase as the mammary cell acquires the 
ability to secrete copious amounts of milk. Similarly, large increases 
in RNA, considered a measure of protein S3mthetic activity, can be ob- 
served in mammary tissue at this time (Denamur, 1974). Preferential 
increases in the activities of lactose synthetase, acetyl-CoA carboxylase, 
and lipoprotein lipase, occur at lactogenesis (Shirley et al. , 1971; 
Baldwin and Yang, 1974; and Convey, 1974). These are believed to be the 
rate limiting enzymes for lactose synthesis, fatty acid synthesis, and 
fatty acid uptake, respectively. Increases in activities of the NADP- 
linked dehydrogenases and acetyl CoA synthetase also were associated 
closely with lactogenesis, although changes were less dramatic (Baldwin, 
1966; Mellenberger et al. , 1973; Baldwin and Yang, 1974; Mellenberger 
and Bauman, 1974a). Thus, the mammary epithelial cells acquire both the 
cellular organelles and the biochemical machinery required for the syn- 
thesis and secretion of milk in a relatively short period of time. 

The timing of lactogenesis varies among species. Extensive bio- 
chemical studies have indicated lactogenesis occurs in the final day 
of pregnancy in the rat (Baldwin, 1966; Baldwin and Yang, 1974), when 
the appearance of a-lactalbumin and lactose was first noted in the tissue 
(Kuhn, 1968). The activity of glucose-6-phosphate dehydrogenase, the 



^N 



-19- 

first enzyme in the pentose-phosphate cycle, also increases during the 
last day of pregnancy (Kuhn, 1968). 

Lactogenesis in the rabbit occurs in two phases. Changes in mammary 
gland KNA:DNA ratios have been demonstrated between day 19 and day 24 
of pregnancy by Denamur (1974), and between days 15 and 24 of pregnancy 
by Mellenberger and Bauman (1974b). In addition, rabbit mammary tissue 
begins to synthesize detectable amounts of lactose (Mellenberger and 
Bauman, 1974b) and fatty acids (Strong and Dils, 1972; Mellenberger and 
Bauman, 1974a) nine to 12 days prepartum (days 19-22 of pregnancy). 
Ultrastructural differentiation occurred by day 21 of pregnancy as noted 
by the development of the rough endoplasmic reticulum and Golgi apparatus 
(Bousquet et al. , 1969). 

No further increases in RNA or metabolic activity, and no further 
change in ultrastructure occurred in rabbit mammary tissue from day 21 
until near parturition. Mammary contents of RNA and DNA, and rates of 
lactose and fatty acid biosynthesis remained constant (Strong and Dils, 
1972; Mellenberger and Bauman, 1974a, b). However, lactogenesis stage 
II, as defined by Fleet et al. (1975), occurred in the rabbit between 
day 29 of pregnancy and day 2 of lactation. This was indicated by in- 
creased RNA, RNA to DNA ratios, and lactose and fatty acid biosynthesis, 
all of which continued to increase in early lactation (Strong and Dils, 
1972; Mellenberger and Bauman, 1974a, b). 

Relatively few biochemical studies of the timing of lactogenesis 
in the cow have been reported. Mellenberger et al. (1973) noted in- 
creased synthesis of lactose and fatty acids (in vitro ) from tissue 
taken at 7 days prepartum compared to 30 days prepartum, and the pat- 
terns of lipids sjmthesized changed from those characteristic of cellular 



-20- 



lipids to those characteristic of milk fat. Kinsella (1975) noted 
similar changes in the rate and pattern of lipid synthesized by bovine 
mammary tissue from days 18 to four prepartum. Activities of several 
enzymes necessary for lactose and fatty acid synthesis also increased 
at this time (Mellenberger et al. , 1973). These studies suggested that 
lactogenesis stage I occurred between day 18 and seven prepartum in the 
cow. Studies by Saake and Heald (1974) and Feldman (1961) reported that 
ultrastructural changes characteristic of lactogenesis had occurred on 
the day of parturition, but that differentiation continued into lacta- 
tion. Enzyme and metabolic flux studies concur that bovine mammary 
tissue continues to increase in the capacity to synthesize milk during 
lactation (Mellenberger et al. , 1973; Kinsella, 1975). Thus lactogenesis 
stage 11 in the cow occurs at parturition, and continues into lactation. 

Endocrine Control of Lactogenesis 

Several reviews have considered the endocrine control of lactogenesis 
in detail (see Denamur, 1971; Convey, 1974; Tucker, 1974; Cowie et al. , 
1980). Early investigations proposed two concepts concerning hormonal 
control of lactogenesis. The first was that substances were present 
during pregnancy (but not at parturition) which were inhibitory to lacto- 
genesis. These inhibitory substances were believed to be ovarian steroids 
and placental hormones. The second concept was that a positive stimulus 
from the anterior pituitary triggered the initiation of lactogenesis. 
This was based on the fact that injections of anterior pituitary ex- 
tracts could induce lactation in pseudopregnant rabbits. 

An enlightening series of experiments by Reece (1939), Folley and 
Young (1941), Meites and Turner (1942a, b) , Desclin (1952), Meites (1954), 



-21- 



and Meites and Sgouris (1954) , established that steroid hormone inhibi- I 

i 
tion and lactogenic complex stimulation are involved in the hormonal [ 

regulation of lactogenesis . In addition, extensive cell and explant 

culture work over the past two decades indicated that the lactogenic I 

complex must include prolactin, a glucocorticoid, and insulin. A dis- j 

cussion of several hormones follows. i 

I 
The presence of insulin is required for mammary tissue to survive :' 

in culture (Elias, 1959), and to obtain a lactogenic response from other ' 

hormones in vitro (Rivera and Bern, 1961). Insulin's role in lacto- i 

genesis is probably only "permissive" in that it is required for glucose 

uptake by the epithelial cell (Mayne and Barry, 1970). ! 

I 

While estrogen administration to animals with well-developed mammary | 

I 

I 

glands initiated lactation (Meites, 1961), this effect may have been due j 

i 

to the ability of estrogen to stimulate the synthesis and secretion of j 

I 

prolactin from the anterior pituitary and /or the ntmiber of prolactin 

receptors in mammary tissue. Estrogen also stimulates the secretion of i 

thyroid stimulating hormone (Topper and Freeman, 1980). Ceriani (1976) [ 

and Bolander and Topper (1979) observed that cultures with estradiol ; 

had augmented casein synthesis, but it is not known whether this effect '. 

was by estrogen itself, or via induction of prolactin receptors in the [ 

mammary tissue (Hayden et al. , 1979). 

Progesterone additions to culture media containing insulin, hydro- 
cortisone, and prolactin blocks RNA and rought endoplasmic reticulum 
formation in rabbit mammary tissue explants (Assairi et al. , 1974). 
Turkington and Hill (1969) demonstrated that progesterone blocked the 
prolactin-induced increase in a-lactalbumin found in tissue cultures. 
Progesterone antagonizes the induction of prolactin receptors by prolactin 



'"*"^ -*''-^-^7/Vrfi^*-— rrr'Hrt>'ii*T»— sa^«.^»i»**i.si^«!:^^l??.'^-f'wr 



-22- 



(Djiane and Durand, 1977), and can competitively inhibit binding of 
glucocorticoids (Collier and Tucker, 1977). Thus, the progesterone 
effect on lactogenesis is an inhibitory one. 

Glucocorticoids are a member of the lactogenic complex. Exogenous 
glucocorticoid administration during mid or late pregnancy can initiate 
lactation in several species with the degree of milk secretion depending 
upon the stage of pregnancy (Denamur, 1971). Ellas (1959) reported that 
corticoids were essential for mammary tissue survival in culture and 
corticoids have been shown to induce formation of rough endoplasmic 
reticulum, casein messenger RNA, and casein (Collier et al. , 1977; 
Topper and Freeman, 1980). It is possible that the role of corticoids 
is to provide the ultrastructural changes necessary for protein synthe- 
sis to occur. The biological effects of adding glucocorticoid to mouse 
mammary tissue can be simulated with spermidine (Topper and Freeman, 
1980), and inhibition of spermidine biosynthesis can prevent the hor- 
monal induction of milk proteins in vitro . Therefore, spermidine may 
be a second messenger for glucocorticoids in mouse mammary tissue. 

It has been shown that growth hormone substitutes effectively for 
prolactin to induce differentiation of mammary tissue in vitro (Rivera 
et al., 1967; Katiyar et al. , 1978). However, little is known about 
the role of growth hormone in vivo as it relates to lactogenesis. It 
is possible that it synergizes with prolactin to induce mammary differ- 
entiation. 

Evidence that prolactin participates in lactogenesis was indicated 
by the depressed milk yields and reduced tissue enzyme activities of 
cattle given ergocryptlne to suppress prolactin secretion during the 
periparturient period (Schams et al., 1972; Akers et al., 1981). It 



■• •- y— >--a m tiM' ^ fii : i<i>-* 



-23- 

also was established by culture studies where addition of prolactin 
enhanced secretory responses of tissues, activities of enzymes, increased 
milk component synthesis, or caused ultrastructural changes character- 
istic of lactogenesis (Mills and Topper , 1970; Delouis and Denamur, 1972; 
Forsyth et al. , 1972; Collier et al. , 1977; Topper and Freeman, 1980). 
However, the mechanism by which prolactin exerts its lactogenic effect 
is unknown (Topper and Freeman, 1980). Falconer and Rowe (1977) demon- 
strated that prolactin activated the extrusion of sodium ions from, and 
the entry of potassium ions into, rabbit mammary epithelial cells. How- 
ever, Bisbee et al. (1979) observed the opposite effect (sodium absorp- 
tion) by adding prolactin to mouse mammary cells on floating collagen 
gels. The possibility prolactin might exert its effect on mammary tissue 
by water and electrolyte movement seems promising in light of its well 
known osmoregulatory effects (Turner and Bagnara, 1976). 

The ability of placental lactogen to inhibit binding of prolactin 
to lactogenic receptors (Shiu et al. , 1973), and to induce lactogenesis 
in hypophysectomized rabbits (Topper and Freeman, 1980) suggests that 
placental lactogen is important for lactogenesis. Placental lactogen 
from some species substitutes effectively for prolactin to induce lacto- 
genesis in vitro (Topper and Freeman, 1980), and in several species is 
found in the circulation at high concentrations during the second half 
of gestation (Blank et al. , 1977). Concentrations in maternal plasma 
may be much lower in the cow, however (Schellenberg and Friesen, 1977), 
and considerable work is needed before its role in lactogenesis in the 
bovine will be clear. 



-24- 



D. Conceptus Hormone Production i 

The placenta in most (and possibly all) species acts as an endo- j 

crine organ and regulates the transport of nutrients from mother to [ 

fetus (Mulay et al. , 1980). Large amounts of both steroid and protein 

hormones are synthesized by the placenta during pregnancy (Simpson and t 

i 
MacDonald, 1981). Estimates indicate that the human placenta at term 

synthesizes one to two grams of placental lactogen per day (Gusseck, i 

1977), meaning that a woman will secrete more placental lactogen in the 

last month of pregnancy than insulin during her entire life (Gusseck, 

1977). Steroid hormones are synthesized and secreted in copious quanti- i 

ties, as well. Unfortunately, the precise roles of these hormones are ;- 

not understood and little is known about how their secretion is regulated ] 

(Porter, 1980). This is unfortunate since considerable evidence sug- ; 

gests that conceptus-derived hormones have important functions in mater- | 

i 
nal recognition of pregnancy, extension of the lifespan of corpora lutea, i 

alteration of maternal metabolism and fetal growth, stimulation of ma- ( 

ternal mammary development and lactogenesis, and induction of parturition. ;. 

One of the fascinating aspects of reproduction is the variety of 

placentation forms among the species (see review by Steven, 1975). ' 

According to Grosser 's Classification (see Perry, 1981) the placental [ 

type characteristic to the cow and the pig is the epitheliochorial type. 

This designation means the chorionic trophoblast comes in close contact 

with the uterine epithelium but does not invade it (Perry, 1981). While 

the pig and cow both have the epitheliochorial type placenta, the pig 

placenta is characterized as the "diffuse" type, while that in the cow 

is termed "cotyledonary" based on the specialized junction of nutrient 






-25- 



exchange between the mother and fetus (Perry, 1981). In the "diffuse" 
placenta the chorion becomes attached to the endometrial wall by inter- 
locking microvilli which effects a tremendous increase in surface area 
between the fetus and mother. Where the chorion comes in contact with 
uterine glands, the chorion arches to create a space for uterine gland 
secretions, called areolae. These areolae are presumed to function as 
areas of exchange for nutrients produced and/or delivered by the uterine 
glands. Gaseous exchange, and possibly exchange of small molecular 
weight nutrients, is accomplished across the villous area. In contrast, 
the only villous connections in the ruminant placenta are at placentomes 
which form adjacent to the uterine caruncles (Perry, 1981). The spaces 
between cotyledons in the cow placenta are characterized by simple ap- 
position of the fetal and maternal epithelia, without interlocking 
microvilli. There also are specialized zones of bovine fetal chorion 
which appose the uterine glands and are, therefore, analogous to the 
porcine areolae (Perry, 1981). 

Differences in placental structure between the pig and the cow may 
be related to differences in placental hormone production by these two 
species. Porcine embryos and extraembryonic membranes are capable of 



diol-17B (Perry et al. , 1973; Fisher et al. , 1981). Along these lines 
Knight et al. (1977) observed lower concentrations of progesterone and 
higher concentrations of estrone and estradiol in uterine vein plasma 
than in radial vein plasma in pregnant gilts. This suggested that por- 
cine conceptuses were metabolizing progesterone to estrogens. Additional 
evidence for porcine conceptus steroid hormone metabolism was provided 
by Chew et al. (1979) and Stoner et al. (1980). These workers observed 



converting radiolabeled androgens and progesterone to estrone and estra- i 






-26- 



significant positive relationships between the number of conceptuses in 
utero and the concentrations of estrone-sulfate in maternal plasma on 
day 30 of gestation. Porcine placental tissue in vitro also synthesized 
estrone and estradiol from radiolabeled androgens (Ainsworth and Ryan, 
1966) . 

There also is evidence that bovine conceptuses have the ability to 
produce estrogens. In 1970 Osinga reported that urinary output of estrone 
in pregnant cows was related to birth weight of the calf (see Thatcher 
et al. , 1980). Eley et al. (1979) observed increased concentrations of 
estrone in allantoic and amniotic fluid which occurred concomitantly 
with increased fetal membrane weights. This was presumed to be due to 
conceptus steroid production. Concentrations of conjugated estrogens 
in maternal plasma increased after day 70 (Eley et al. , 1979) and after 
day 72 (Robertson and King, 1979) and were presumably due to conceptus 
production. Eley (1980) demonstrated that the bovine conceptus could 
synthesize estrogens from steroid precursors in vitro. 

Studies in the pig and cow indicated that ovariectomy during preg- 
nancy caused abortion which suggests that the bovine and porcine con- 
ceptus (es) do not produce quantities of progesterone adequate to sustain 
pregnancy. 

Placental lactogen was hypothesized to exist in the cow because 
coculture of cotyledon and mammary tissue explants produced a lactogenic 
response (Buttle and Forsyth, 1976). Similar attempts to produce a 
lactogenic response with porcine placenta were unsuccessful, however, 
and led to the conclusion that there was no placental lactogen in the 
pig (Forsyth, 1974). Subsequently, there have been five reports of the 
purification of bovine placental lactogen (Bolander and Fellows, 1976; 



L—— i.iifc.- v-.--^ -- -'_-,- ^1 '---i--^. ,'ilf-r-'.~-'"^'"— ?^ r ■ '•-■•^~T'Ti-.-» 



-27- 



Roy et al., 1977; Hayden and Forsyth, 1979; Bremel et al. , 1979; and 
Beckers et al., 1980). Unfortunately, discrepancies existed between 
these publications with regard to molecular weight estimates of the 
hormone (22,000 to 60,000). Large discrepancies in the literature also 
existed with respect to the concentrations of bovine placental lactogen 
in maternal plasma. Using a homologous radioimmunoassay with their 
purified bovine placental lactogen preparation, Bolander et al. (1976) 
reported that concentrations were greater than 1100 ng/ml in dairy cows 
during late gestation. In contrast, Kelly et al. (1973), Buttle and 
Forsyth (1976), and Schellenberg and Friesen (1981) using bioassay or 
radioreceptor assay indicated that placental lactogen concentrations in 
the pregnant cow were very low or undetectable. This may require a re- 
evaluation of the function of bovine placental lactogen. Human placental 
lactogen has well known metabolic effects in addition to its role in 
stimulating mamogenesis (Josimovich and Archer, 1977). Kaplan and 
Grumbach (1974) showed that acute administration of placental lactogen 
caused a marked increase in maternal plasma free fatty acids, presumably 
to spare carbohydrates for fetal use. Placental lactogen in rats and 
mice sustains the production of estrogen and progesterone from corpora 
lutea, thereby exhibiting a role in steroidogenesis (Josimovich and 
Archer, 1977). 

Another of the possible roles for placental lactogen may be that 
of osmoregulation. Evidence suggests that prolactin added to the fetal 
side of the human term amnion significantly decreased water diffusion 
from fetal to maternal side (Leontic et al., 1979). Addition of a speci- 
fic prolactin receptor antibody completely abolished the effect of pro- 
lactin, as did the addition of ouabain (Leontic et al. , 1979). Bazer 



i-««**— .;ii^' i.-^ ,-iK.ti» 5*!n« <y*iL 



-28- 

et al. (1981) conducted a series of experiments to examine the effect 
of human placental lactogen, prolactin, and steroid hormones on trans- 
port properties of the porcine chorioallantois. Human placental lactogen 
elicited responses in short circuit current and potential differences 
across the chorioallantois which suggested it may play a role in placen- 
tal active transport processes (Bazer et al. , 1981). It also was noted 
that ergocryptine administration to gilts resulted in decreased allan- 
toic fluid volumes in fetuses on day 30 of gestation. Thus, prolactin 
may function in preventing water movement from fetus to mother across i 

the porcine chorioallantois (Bazer et al. , 1981) as it does across the 
human amnion (Leontic et al. , 1979). It also was interesting that 
periods of allantoic fluid expansion were associated with increasing 
estrogen to progesterone ratios in the mother. Estrogen has been shown 
to induce the prolactin receptors in mammary tissue (Hayden et al. , 
1979), and lactogenesis occurs during a period when the estrogen to 
progesterone ratio is increasing. Perhaps a primary role of prolactin 
and placental lactogen during pregnancy is to regulate active transport 
across placental membranes. 



-■ny - <-.i|i»*— ^-I-j^H-.'-l^'-IMnitT- 



CHAPTER III 

CHARACTERIZATION OF MAMMOGENESIS AND LACTOGENESIS 

IN THE GILT 



A. Nucleic Acid, Metabolic, and Histological Changes in Gilt 
Mammary Tissue During Pregnancy and Lactogenesis 



Introduction 



Prior to determining the influence of conceptus units on mammo- 
genesis and lactogenesis one must first have a true understanding of 
these processes. In particular, defining mammogenesis and lactogenesis 
in the gilt includes defining the structural changes that occur in 
the mammary gland during mammogenesis and lactogenesis, characterizing 
changes in concentrations of hormones and rates of metabolic activity 
of mammary tissue during this time, examining the relative utilization 
of different substrates, and defining specifically when in pregnancy or 
lactation these changes occur. 

Although mammary growth and lactogenesis have been fairly well 
characterized in rats (Chatterton et al. , 1975), rabbits (Strong and 
Dils, 1972; Mellenberger and Bauman, 1974a, b), and cows (Mellenberger 
et al. , 1973), comparatively little is known about these processes in pigs. 
Hacker and Hill (1972) examined mammary gland DNA and RNA on days 25, 50, 
and 100 of pregnancy in gilts, and Turner (1952) described the gross 
histological changes that occurred during pregnancy. However, no 
studies have been conducted to integrate changes in cell concentration, 



-29- 






-30- 



metabolic activity, and maniraary tissue structure during pregnancy and 
lactogenesis. Several investigators have studied sow mammary tissue 
with respect to various metabolites and biochemical pathways utilized 
(Linzell et al. , 1969; Spincer et al. , 1969; Bauman et al. , 1970), but 
no studies have been conducted to examine changes in the rate of metabo- 
lism during lactogenesis. Objectives of this study were (1) to define 
changes in mammary gland DNA, RNA, and histology (indices of mammogene- 
sis) during gestation, and (2) to examine the relationship between 
lactogenesis in gilts and changes in RNA, RNA to DNA ratio, histology, 
and in vitro rates of substrate oxidation and lipogenesis in short-term 
inc libation. 

Materials and Methods 

Experimental design and treatments . Twenty-one crossbred gilts of 
similar age, weight, and genetic background were assigned randomly (three 
per group) to undergo a single mammary biopsy on day 30, 45, 60, 75, 90, 
105, or 112 of pregnancy. Mammary tissue biopsies of pregnant animals 
were performed in conjunction with Charles Ducsay during his studies on 
iron transport in the fetal pig (Ducsay, 1981). Six additional gilts were 
randomly assigned to undergo a mammary biopsy on the day of parturition 
(day 115 ± 1, three gilts) or the fourth day of lactation (day 4, three 
gilts). These biopsies were performed by H.N. Becker (College of Veterinary 
Medicine, University of Florida). At biopsy, gilts were anesthetized 
with sodium thiamylal and anesthesia was maintained with methoxy 
flurane (Pittman-Moore) . In all 27 gilts, a single mammary tissue 
biopsy of approximately 6 grams (g) was taken from the second most 



t r.--b»i^'i<»^i^-*j**,',^fwri-->l|-. 



-31- 



posterior inguinal gland on the right side. Mammary biopsies on the 
day of parturition were performed 4 to 12 hours (hr) after the last pig 
was born. Approximately 2 g of mammary tissue were placed immediately 
in buffered neutral formalin solution for fixation. Two grams of mammary 
tissue were frozen at -20 C for subsequent nucleic acid analyses, and 
the remaining tissue was placed quickly in ice-cold, isotonic tris- 
sucrose solution, pH 7.3, until used for metabolic activity studies. 

Histological procedures . Mammary tissues designated for histologi- 
cal examination were fixed in buffered neutral formalin for 48 hr. Sub- 
sequently, samples were dehydrated and embedded in paraffin (Paraplast 
Tissue Embedding Medium; Lancer, St. Louis, MO). After the paraffin 
blocks were hardened in a freezer, 5 to 7-u thick sections were made 
with a Spencer-American Optical microtome (American Optical Co., Buf- 
falo, NY). After drying, thick sections were stained with Harris' Alum 
Hematoxylin (McClung Jones, 1966) and counter-stained with .5% eosin in 
alcohol (Sanders, 1972). Histological photomicrographs were taken with 
a Leitz microscope at X150 magnification. 

Metabolic rate study . Slices of mammary tissue (three replicates 
per gilt) 0.5 mm in thickness and weighing 100 to 180 mg were prepared 
with a Stadie-Riggs hand microtome (Stadie and Riggs, 1944) and placed 
in 25-ml Erlenmeyer flasks with 3 ml Krebs-Ringer bicarbonate buffer 
(pH 7.3). The buffer also contained 10 mM acetate, 10 mM glucose, 100 
mil insulin/ml, and 1 liCi radioactive substrate/ml [either 2- C-acetate 
(New England Nuclear, Boston, MA) or U- C-glucose (ICN Pharmaceutical, 
Irvine, CA) ] . These conditions were based on factors affecting bovine 
mammary tissue incubations (Bauman et al., 1973). Tissues then were 
incubated for 3 hr in a Dubnoff metabolic water bath at 37 C in an 






-32- 



atmosphere of :C0„ (95:5). Substrate oxidation and incorporation 
into fatty acids were measured according to methods described by Bauman 
et al. (1970). A preliminary study was conducted to establish that C0„ 
production and fatty acid synthesis rates were linear for lactating 
porcine mammary tissue over a 3 hr incubation period. 

Nucleic acid and dry fat-free tissue analysis . Approximately . 5 g 

of mammary tissue was cut into 20 to 25 pieces and added to 10 ml ice- I- 

I 

cold 10 m^I tris-HCl buffer, pH 7.5. Mammary tissue was fully disrupted | 

I- 

with three 15 s bursts of a Polytron homogenizer (Brinkman Istruments, f 

Westbury, NY) at half speed. Heavier cell components (including nuclei) [ 

r 

then were isolated by centrifugation of the homogenate at 7,000 g for 

10 min. Water and lipid were extracted from the pellet using 5 ml of ; 

absolute ethanol and then 5 ml of ether. The pellet remaining after j 

ether extraction was air-dried for determination of a dry, fat-free i 

tissue weight. Pellets then were extracted twice with ice-cold 10% ': 

i 

trichloroacetic acid (TCA) . Excess TCA was removed by washing with i 

ice-cold absolute ethanol saturated with sodium acetate. The RNA and ; 

DNA were separated and quantified according to methods described by 

I 
( 

Tucker (1964), as adapted from Schmidt and Thannhauser (1945). ' 

f 
Statistical analyses . All data were analyzed by least-squares i 

analysis of variance according to the General Linear Models procedures [ 

of the Statistical Analysis System (Barr et al. , 1976). Linear reac- 

tion rates for the mammary tissue slice incubation study were estab- ; 

lished by examining polynomial effects of hour when used as a continuous 

variable. Effects of day of pregnancy on mammary DNA concentration, 

RNA concentration, RNA:DNA ratio, and percentage dry, fat-free tissue 

were examined. Overall effects of day of pregnancy and gilt were 



-33- 



examined for in^ vitro metabolic rate parameters including acetate, and 
glucose oxidation and incorporation into lipid. In addition, orthogonal 
single degree of freedom comparisons (see table III-l for comparisons) 
were made to test variation between days. Since each gilt was nested 
within day, gilt-within-day mean squares served as the error term for 
days. 

Results and Discussion 

Histological development . Histological changes in gilt mammary 
tissue during mammogenesis and lactogenesis are shown in photomicro- 
graphs A through D in figures III-l and III-2. All photomicrographs 
are at X150 magnification. Histological appearance on days 30 and 45 
was similar. Plate A of figure III-l is a representative photomicro- 
graph of tissue from gilts 45 days pregnant. At this time, cell types 
present included those in connective and adipose tissue and cells 
comprising large and small ducts that proliferate into the mammary gland 
fat pad. The two-layered epithelium of mammary ducts was distinguish- 
able from the slender elongated nuclei of the stromal cells. Increased 
branching of the duct system with little or no development of end buds 
is similar to Turner's (1952) whole mount observations of day 30 gilt 
mammary tissue. By day 60 of pregnancy (plate B, figure III-l), further 
invasion of epithelial cells into stromal tissue was apparent, indi- 
cating further duct growth, and lobule formation had begun. Plate C of 
day 75 tissue demonstrated a clear increase in mammary epithelial cell 
concentration that occurred after day 60. A large increase in number 
of alveoli was apparent by day 75, and these were located in well-defined 



Figure III-l. Histological photomicrographs of porcine mammary tissue 
at days 45 (A), 60 (B) , 75 (G) , and 90 (D) of pregnancy. 
Magnification X150. 



r-^-"-ill;^a^ ^* - ^ -- ? »^ r~ :?i.^ f .■ v^ ^.i^^ea^gi. - 



-35- 



v::-<r^* 






u*i 



"»,* 



l^*^A 






-*-"H'^ilM?«3i^v-^r 



-36- 



lobules with rapidly decreasing areas of adipose and connective tissue 
surrounding them. However, secretion was not readily evident in lumina 
of these alveoli. 

At day 90 (plate D, figure III-l) , very little adipose tissue re- 
mained. A single band of connective tissue was evident in the center 
of the photomicrograph, with lobules on either side. Although growth 
of the gland appeared complete histologically, there was only sparse 
accumulation of colostrum in alveolar lumina. 

Between days 90 and 105 (plate A, figure 1II-2) of pregnancy, a 
dramatic change in appearance of the tissue was evident. Abundant 
secretion had accumulated in the lumina of alveolie by day 105. Eosino- 
philic staining of this secretion suggested the presence of protein in 
large quantities. Lipid droplets also were apparent within secretory 
cells and in the lumina of the alveoli. Thus, between days 90 and 105 
of pregnancy, differentiation of gilt mammary tissue from the nonlactating 
to the lactating state had begun. Estrogens, which cause immunoglobulin 
movement from blood into the mammary gland (Smith et al., 1971), in- 
crease rapidly at this stage of pregnancy (Robertson and King, 1974; 
Knight et al., 1977), with a concurrent decrease in progesterone con- 
centrations. Turner (1952) also reported the distension of alveolar 
lumina in mammary tissue of day 105 pregnant gilts. 

By day 112 (plate B, figure III-2) , extreme distension of alveoli 
occurred as secretory products accumulated in the lumen. The entire 
apical portion of epithelial cells on day 112 appeared to be composed 
of lipid droplets. This was not apparent in tissue obtained postpartum 
(plates C and D, figure 1II-2) in that lipid was not accumulating in 
cells but was transported out into the lumen. Thus, in tissue obtained 



Figure III-2. Histological photomicrographs of porcine mammary tissue 
at days 105 (A) and 112 (B) of pregnancy and days 1 (C) 
and 4 (D) of lactation. Magnification X150. 



-38- 



















^^^.■■^pr ». 








-39- 



postpartum secretion of product was fully coupled with synthesis. The 
incomplete differentiation of the secretory process prepartum was similar 
to that suggested by Chatterton et al. (1975). 

Mammary tissue collected on day of parturition of gilts was ex- 
amined by Turner (1952) , Hacker (1970) , and in the present study (plate 
C, figure III-2) . All results indicated that alveoli appeared fully 
functional. Suckling activity by the litter had removed colostrum from 
most alveoli, as evidenced by decreased eosinophilic staining, so that 
the secretion had the appearance of normal milk with numerous lipid 
droplets (plate C, figure III-2) . Also, lipid previously observed in 
the apical end of each cell was absent in those cells in tissue from 
day of parturition, suggesting that the process of secretion was fully 
coupled with the process of synthesis. Plate D (figure III-2) depicts 
mammary tissue from a gilt on day 4 of lactation. Accumulation of milk 
with many lipid droplets in the Ixomina suggests a high rate of metabolic 
activity. Alveoli were highly differentiated at this time, with 
epithelial cell nuclei tightly pressed against the basal borders of 
the cells. Very few adipose or other connective tissue cells were 
present at this time, and secretory epithelium occupied most of the 
gland . 

Nucleic acids . Concentrations of DNA in tissue biopsies during 
pregnancy to day 4 of lactation (day +4) and changes in percentage of 
mammary tissue wet weights (WMT) which corresponded to dry, fat-free 
tissue (DFFT) are presented in figure III-3. Although the concentration 
of DNA per gram wet mammary tissue (milligrams DNA per gram WMT) appeared 
to increase from day 30 to 45 of pregnancy (.211 vs .827 mg DNA per g 
WMT), this increase was not significant statistically (see table III-l) 





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due to the large variation among gilts. The amount of DNA per g WMT 
remained at approximately .8 mg through day 75, when a burst of epi- 
thelial cell proliferation occurred and near maximum concentrations of 
DNA (3.8 mg per g WMT) were attained by day 90 of pregnancy. The in- 
crease in DNA observed on day 90 agreed with histological changes noted 
in this tissue in figures III-l and III-2. On day 112 of pregnancy and 
>n the day of parturition (day 115 of pregnancy or day +1 of lactation), 
there was a slight decrease (P < .04) in concentration of DNA compared 
to day 4 concentration, probably reflecting cellular hypertrophy and 
accumulation of water in the mammary alveolar lumina with the build-up 
of colostrum. Colostrum accumulation was apparent in photomicrograph B 
(figure III-2) of day 112 mammary tissue. By day 4 of lactation, pig- 
lets had removed milk accumulated before birth, and DNA concentrations 
increased to concentrations observed before the onset of lactogenesis. 
The percentage of DFFT (figure III-3) showed a pattern similar to 
that of mg DNA per g WMT, suggesting that the DFFT was composed primarily 

f mammary epithelium and its stroma. Thus, as epithelial and suppor- 
tive connective tissue invaded the fat pad of the mammary gland, a 
decline in lipid concentration occurred and was reflected by increased 
DNA concentration and percentage DFFT. Also, there was more DFFT in 
mammary tissue after day 75 of gestation (P < .01) than before that 
time (comparison A, table III-l). There was also a slight depression 
(P < .09) in percentage DFFT on days 112 of pregnancy and 1 of lacta- 
tion, similar to that observed for DNA (figure III-3 and table III-l). 

Hacker and Hill (1972) measured total amount of lyophilized fat-free 
tissue (LFFT), DNA, and RNA in mammary glands of gilts on days 25, 50, and 
100 of pregnancy. He observed that amount of LFFT did not change between 



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-44- 



days 25 and 50, but increased sixfold between days 50 and 100. In the 
present study, percentage DFFT did not change significantly from day 30 
through day 75, but increased more than fourfold by day 90 (figure 
III-3) . Collectively, histological and DNA concentration changes in 
gilt mammary tissues indicated increased tissue growth between days 75 
and 90 of pregnancy following completion of placental growth (Knight 
et al., 1977). Maximum cell concentrations were attained by day 90, 
after which the process of lactogenesis began. 

Concentrations of RNA did not change from day 30 through 75 of 
pregnancy, but increased between days 75 and 90 (figure III-4, table 
III-l) . Concentrations of RNA through day 90 of pregnancy paralleled 
mammary epithelial cell numbers, as was apparent by the absence of 
change in RNA:DNA (figure III-4) . After day 75, RNA increased steadily 
through the fourth day of lactation (table III-I, figure III-4) . There- 
fore, in contrast to DNA, RNA continued to rise through day +4, and 
was indicative of cellular hypertrophy. 

The RNA: DNA ratio, an expression of the protein synthetic activity 
of each cell, remained relatively stable between days 30 and 90 of 
pregnancy (figure III-4, table III-l). However, from day 90 of preg- 
nancy through day +4, there were progressive increases in RNA:DNA ratio 
to .8, 1.2, 1.8, 2.1, and 2.5 on days 90, 105, 112, +1, and +4, respec- 
tively. Presumably this signaled initiation of lactogenesis (Denamur, 
1974) , since each secretory cell acquired the capacity first to secrete 
colostrum and later to synthesize and secrete milk. This is evident in 
photomicrograph B (figure III-2) , from tissue biopsied on day 105, 
when significant amounts of colostrum-like material in alveolar lumina 
first were noted. Hacker (1970) estimated RNA:DNA ratio to be 1.6 on 



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day 110; the estimate for day 112 (figure III-4) in this study was 1.8. 
Data from this study and that of Hacker (1970) indicated RNA:DNA ratio 
to be 2.1 on day of parturition. The RNA:DNA ratio subsequently in- 
creased to 2.4 on day 2 of lactation (Hacker, 1970) and, in this study, 
2.6 on day 4 (figure III-4) . Based on results for RNA:DNA ratio, as 
well as RNA, DNA, and histological changes, the period between days 90 
and 105 is associated with the onset of lactogenesis in the gilt. Thus, 
as with the rabbit (Strong and Dils, 1972; Mellenberger and Bairaian, 
1974a, b) and cow (Mellenberger et al., 1973), the process of lactogenesis 
in the pig occurred in two phases and was initiated well before parturi- 
tion. 

Metabolic changes . To establish that oxygen and substrates were 
not limiting over a 3-hr period in the incubation system utilized in 
this study, mammary tissue from a single lactating sow was incubated 
for 1, 2, or 3 hr (three incubations per radioactive substrate per hr) . 
The quantities of acetate and glucose oxidized or incorporated into fatty 
acids after different lengths of incubation were determined and are 
presented in table III-2 and figure III-5. In figure III-5, the rates 
of metabolism of acetate and glucose were combined to form a comprehen- 
sive two-carbon unit (TCU) oxidation or incorporation (where nmoles 
acetate plus 3 x nmoles glucose equals nmoles TCU) . This presents a 
cumulative measure of acetate and glucose utilization which best illus- 
trates the total amount of carbon oxidized or incorporated into fatty 
acids by tissue from radiolabeled acetate and glucose substrates. 
Significant first order equations were fit for each of the variables 
and indicated that porcine mammary tissue metabolized these substrates 
in a linear fashion over the 3-hr incubation period (table IH-2) . 



■' ^y-zr-T-^tr"''''"- 



-48- 



Table III-2. EFFECT OF INCUBATION TIME ON SUBSTRATE OXIDATION AND 
INCORPORATION INTO FATTY ACIDS^ 

Hours of incubation 



Acetate oxidation 
Glucose oxidation 



140 
217 



282 
401 



529 
658 



Acetate incorporation 
Glucose incorporation 



760 

45 



1,266 
90 



2,066 
134 



All units are in nmoles substrate per 100 mg tissue and represent the 
mean of three incubations. 

Described by linear equation Y = -82.8462 + 198.6923X where Y = nmoles 
acetate oxidized per 100 mg tissue and X = incubation time in hr 
(r2 = .84, P < .002). 

"Described by linear equation Y = -15.8888 + 220.6666X where Y = nmoles 
glucose oxidized per 100 mg tissue and X = incubation time in hr 
(R^ = .87, P < .001). 

i 

Described by linear equation Y = 27.4103 + 664.5128X where Y = nmoles 
acetate incorporated into fatty acids per 100 mg tissue and X = incu- 
bation time in hr (r2 = . 92 , P < .0001). 

""Described by linear equation Y = .7777 + 44.5000X where Y = nmoles 
glucose incorporated into fatty acids per 100 mg tissue and X = incu- 
bation time in hr (R^ = .86, P < .001). 



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Rates of oxidation of acetate and glucose by mammary tissue are 
summarized in table III-3. Rates of oxidation and incorporation into 
lipid were expressed on a per 100 mg WMT basis as well as on a per 
microgram DNA basis (to adjust for the number of cells present). There 
was a marked increase in rate of acetate oxidation per 100 mg WMT be- 
tween the mean value on days 30 and 45 versus the mean value on days 
60 and 75 of pregnancy (P < .05) as the rate increased from 312 nmoles 
acetate per 3 hr incubation on day 45 to 1,585 nmoles on day 60 (table 
III-2). This corresponded to histological evidence (figure III-l) of 
coincident mammary epithelial invasion of the mammary fat pad, and sug- 
gested that lipolysis of cellular triglycerides probably was occurring. 

Rate of acetate oxidation was low from day 90 through day +1 
(table III-3) . Rate of acetate oxidation also appeared slightly higher 
on day +4 than on days 112 and +1, but this difference was not statis- 
tically significant (table III-l). 

Patterns of glucose and acetate oxidation by mammary tissue during 
pregnancy and lactogenesis were different (table III-3) . Glucose oxi- 
dation per 100 mg WMT increased from 217 nmoles per 3 hr on days 30 and 
45 to more than 600 nmoles on days 60 and 75; however, this increase 
was not statistically significant (table III-l, contrast D) . Activity 
was low between days 90 and 112 of pregnancy. On the day of parturition 
(+1), glucose oxidation reached midpregnancy rates and, in the ensuing 
4 days increased sixfold. Specific contrasts indicated that rate of 
glucose oxidation by 100 mg WMT was greater (P < .05) on days 112, +1, 
and +4 than on days 90 and 105, and greater (P < .01) on day +4 than on 
days 112 and +1. Thus, the rate of mammary tissue glucose oxidation 
was relatively low until the final days of pregnancy. Presumably at 



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-53- 



that time stage II lactogenesis imposed increased energy demands and 
increased glucose oxidation (table III-3) . 

Relative contributions of acetate and glucose to the C0„ measured 
indicated that glucose contributed 53 to 68% of the carbon from 30 to 
75 days of pregnancy. However, only 25 to 40% was contributed on days 
90, 105, and 112 of pregnancy. This apparently reflected the diabeto- 
genic effect of pregnancy observed in most mammalian species (George 
et al. , 1978), and represented decreased glucose utilization per gram 
of maternal tissue of the pregnant gilt to support the tremendous rate 
of fetal growth which occurs at this time (Knight et al. , 1977; Bauman 
and Currie, 1980). On the day of parturition, the uterine demand for 
glucose was greatly diminished, and mammary glucose oxidation Increased, 
accounting for 85% of CO^ measured (table III-3) and 92% on day +4. In 
two perfusion studies of lactating sow mammary glands Linzell et al. 
(1969) demonstrated that glucose accounted for 13 and 40% of the CO 
measured, whereas acetate contributed only 2%, The ratio of acetate to 
glucose oxidation they observed (Linzell et al. , 1969) was similar to 
those observed in the tissue incubations from the current study. Bauman 
et al. (1970) observed that glucose accounted for only 35% of the CO 
measured in mammary incubations from sows in late lactation; 4 to 7 
weeks. These rates corresponded to those observed on days 90 and 105 
of pregnancy in the current study. 

Rates of substrate oxidation on a per pg DNA basis are in table 
III-3. Five to 8 nmoles of acetate were oxidized per ug of DNA per 
3 hr on days 30 and 45 of pregnancy. By day 60, the gland oxidized 
over 22 nmoles per 3 hr, and on day 75, 13.5 nmoles per 3 hr per pg 
DNA. A specific comparison (table III-l) indicated that acetate 






-54- 



oxidation per yg DNA was greater (P < .01) on days 60 and 75 than on 
days 30 and 45. Activity decreased precipitously by day 90 and remained 
below 3 nmoles per yg DNA per 3 hr through day 4 of lactation. Acetate 
oxidation per yg DNA was greater (P < .01) before than after day 90 of 
pregnancy. Oxidation of glucose per yg DNA followed a similar pattern, 
except it was more variable and appeared to increase on the fourth day 
of lactation (tables III-l and III-3) . Expressing metabolic data on a 
DNA basis versus a wet weight basis results in a different pattern of 
activity (table III-3) . Rate of acetate and glucose oxidation per yg 
DNA on day +4 was only a fraction of that activity on day 60. In con- 
trast, substrate oxidation per 100 mg of wet tissue was threefold greater 
on day 4 of lactation than at midpregnancy. It is obvious that the 
oxidation rate of the entire mammary gland was greatest at the end of 
pregnancy and during lactation, when maximum numbers of cells were 
present. Surprisingly, the oxidation rate of individual mammary gland 
cells was greatest during midpregnancy, when growth of the mammary 
glands was occurring. This may represent contribution of adipocyte 
lipolysis during mammary invasion of fat pad. 

Data in table III-3 illustrate changes in the rates of incorpora- 
tion of acetate and glucose into lipid by gilt mammary tissue on a wet 
weight basis. Acetate was incorporated at extremely low rates from day 
30 through day 90 of pregnancy (206 nmoles or less per 100 mg per 3 
hr) . On day 105, the rate of acetate incorporation increased over two- 
fold to 451 nmoles per 100 mg per 3 hr, and subsequently increased to 
618, 1,947, and 7,863 nmoles on days 112, -1-1, and -t-4, respectively. 
Changes in acetate incorporation reflect onset of lactogenesis , which 
was initiated around day 105 and then rapidly progressed in the final 






-55- 



days of pregnancy and during early lactation. Rate of lipogenesis from 
glucose expressed on a wet weight basis followed a pattern similar to 
that of lipogenesis from acetate (table III-3) . The rate remained be- 
low 41 nmoles from day 30 through 112 of pregnancy. On day of parturi- 
tion, glucose incorporation increased over 13-fold at 159 nmoles per 
100 mg per 3 hr, and this rate subsequently increased to over 1,100 by 
day +4 (tables III-l, III-3) . This dramatic change in mammary metabolism 
occurred in a short period of time as lipogenesis from glucose was 
93-fold greater on day +4 than on day 112 of pregnancy. 

Relative utilization of acetate and glucose for lipid synthesis 
by mammary tissue slices during incubation changed over the course of 
mammary growth and lactogenesis, but less than did their utilization 
for oxidative purposes. On day 30 of pregnancy, acetate accoiinted for 
46% of the carbon incorporated into lipid during incubation. The rela- 
tive contribution of acetate steadily increased until the final 3 days 
of pregnancy, when approximately 95% of the carbon atoms incorporated 
into lipid were of acetate origin. With parturition and onset of milk 
production, glucose contributed slightly more carbon to lipid synthesis, 
but on day +4, acetate still contributed 70% of the carbon for this pur- 
pose. While acetate was, in general, a greater carbon donor for lipo- 
genesis than glucose, there was still an apparent mammary adaptation to 
utilize minimal amounts of glucose at the end of gestation when fetal 
glucose demands were greatest. 

Rates of incorporation of substrate into lipid on a per yg of DNA 
basis are in table III-3. Rates of acetate incorporated into lipid on 
days 30, 45, 60, and 75 of pregnancy were 1.3, 1.5, 2.5, and 2.5 nmoles 
per yg DNA per 3 hr, respectively (table III-3) . Although acetate 



-56- 



incorporation appeared to decline between day 75 and 90 of pregnancy, 
average rate of incorporation after day 90 was greater (P < .05) than 
before da^^ 90 (table III-l) . From day 90 of pregnancy and on into 
lactation, mammary cells incorporated acetate at increasing rates 
(table III-3) . On the day of parturition, rate of acetate incorpora- 
tion was two- to threefold greater than the rate at midpregnancy , and 
by day +4, it had Increased to 19.1 nmoles per \xg DNA per 3 hr (table 
III-l, comparisons F and H) . Mean rate of glucose incorporation into 
lipid before day 90 of pregnancy did not differ from that after day 90 
(tables III-l, III-3) . However, rates of lipid synthesis from glucose 
by mammary cells on days 90 and 105 of pregnancy were lower (P < .05) 
than on subsequent days. Lipogenesis increased in the final days of 
pregnancy and surpassed midpregnancy rates by the fourth day of lacta- 
tion (day +4 greater than day 112 and +1, P < .01). It was apparent 
that glucose had not been utilized appreciably by mammary tissue of the 
gilt for fatty acid synthesis until the final 3 days of pregnancy. 

Figure IH-6 presents rates of TCU oxidation and TCU incorpora- 
tion over the course of gestation and early lactation. The TCU oxida- 
tion in early pregnancy reflected oxidation of glucose (table III-3, 
figure III-6) more than it did oxidation of acetate. There were small 
but nonsignificant changes in TCU oxidation throughout most of preg- 
nancy (table III-l). Oxidation rate was higher (P < .10) on days 112 
and +1 than on days 90 and 105, and further increased on day +4 (table 
III-l, P < .01) to three times the rate observed at midpregnancy. The 
TCU incorporation per 100 mg WMT reflected the same pattern as acetate 
or glucose, with very low rates of lipogenesis through day 105 of preg- 
nancy (figure III-6) . The TCU incorporation was greater (P < .01) on 



'.i>*»r»..n)^..-— .^i^.: —=f\t 



to CO 




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;*Vfc?«-<A*:.r'-i^»«-~a«,^^--.j— -l'i.^-w>a:.-*tv.(>-T*:f'Jl'_. .-jfrriP •"- 



-58- 



H8/11NM BW OOL/QHZiaiXO ROl SHIOIAJN 




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-59- 



days 112, +1, and +4 than on days 90 and 105, and Increased (P < .01) 
further from days 112 and +1 to +4 (table III-l). 

On days 30, 45, 60, 75, and 90 of pregnancy, four to 10 times as 
much total carbon from acetate and glucose was oxidized as incorporated 
into lipid (figure III-6) . Relative utilization of substrate oxidized 
versus incorporated decreased to a 2:1 ratio on days 105 and 112 of 
pregnancy, and to a 1:1 ratio by the day of parturition and onset of 
extensive milk secretion. 

Several investigators have examined rates of uptake of plasma con- 
stituents and their utilization by lactating porcine mammary tissue for 
milk synthesis (Linzell et al., 1969; Spincer et al., 1969; Bauman et 
al., 1970). All investigators found considerable uptake and utiliza- 
tion of glucose by the sow mammary gland, and relatively low, but con- 
sistent utilization of acetate. Linzell et al. (1969) and Spincer et 
al. (1969) estimated that 50 and 31% of glucose entering the mammary 
circulation was taken up, respectively. Spincer et al. (1969) estimated 
that the mammary gland removed 46% of the acetate in blood, but due to 
the low concentration of acetate in plasma of the pig, this quantity 
was small compared to glucose. Linzell et al. (1969) demonstrated that 
infused acetate was utilized for oxidation and milk fat synthesis, and 
Bauman et al. (1970) found that in the presence of both acetate and 
glucose, sow mammary tissue preferentially utilized acetate for fatty 
acid synthesis. In this study, rate of acetate oxidation per 100 mg 
WMT increased fivefold from day 45 to 60 of pregnancy, then progres- 
sively declined to rates characteristic of early pregnancy by the day 
of parturition (table III-3) . There was an increase in activity on 
day +4, but tissue remained less active than day 60 tissue on a wet 






-60- 



weight basis. In contrast, glucose oxidation rate increased only 
slightly on days 60 and 75 of pregnancy, whereas CO production from 
glucose increased markedly after day 112 of pregnancy (tables III-l, 
III-3) . Surprisingly, mammary tissue generated less energy from these 
substrates on the day of parturition (+1) than during midpregnancy. 

When acetate oxidation was expressed per microgram of DNA, the 
trend differed from that observed when data were expressed on a wet ^ 

weight basis. Acetate oxidation per cell was greater (P < .01) on days 
30 through 75 of pregnancy than on the subsequent days observed (tables 
III-l, III-3) . The same was true for glucose oxidation per cell 
(P < .10, table III-3) . In addition, total oxidation of acetate and 
glucose on day +4 was only a fraction of that observed on day 60 of 
pregnancy. Therefore, mammary epithelial cells undergoing cell division 
utilized more acetate and glucose to generate energy than did fully 
lactating cells. Perhaps this was necessary for the mammary epithelliam 
to mobilize the fat pad and undergo hyperplasia. 

Rates of acetate incorporation into lipid by mammary slices re- 
mained low until lactogenesis began (between days 90 and 105 of preg- 
nancy) and then increased rapidly (table III-3) . Increased rate of 
lipogenesis from glucose was even more dramatic, since the rate on day 
+4 was 93-fold greater than on day 112 of pregnancy. 

Relative contributions of acetate and glucose for lipid synthesis 
were determined from data listed in table III-3. While acetate con- 
tributed 46% of the carbon incorporated into lipid on day 30 of preg- 
nancy, the contribution increased steadily during pregnancy to account 
for 95% of lipid carbon on day 112. Therefore, the share of carbon 
contributed by acetate for lipogenesis increased with advancing pregnancy 



-.■^.)»-"-t,i^iw^>^."«<w..*<'>~fi-.i» —-■»...- - -^- V<-^ — ,v "- tii.i - . -i *-: .» - •-•>. il'^'— -M^.-^"- 



-61- 



as the uterine and fetal demand for glucose increased (Knight et al. , 
1977). With parturition and lactation, acetate still contributed more 
than twice as many carbon atoms as glucose for fatty acid synthesis, 
as ratios of acetate to glucose were 24:1, 16:1, 4:1, and 2.3:1 on days 
105, 112, +1, and +4, respectively. 

On a DNA basis, rates of acetate or glucose incorporated were not 
as high on days 60 and 75 of pregnancy as their oxidation rates (table 
III-3) . However, acetate incorporation was greater than acetate oxida- 
tion during lactation (table III-3) . 

Total nanomoles of substrate oxidized compared to the amount in- 
corporated into lipid (figure III-6) revealed that the ratio changed 
from a peak of 10:1 in proliferating mammary tissue to 1:1 in fully 
differentiated lactating tissue. This shift in the fate of acetate and 
glucose probably reflects the change in cell-types present as the mammary 
epithelium grows into and replaces the mammary fat pad. The adipocytes 
in the gland in early and midpregnancy probably oxidize more substrate 
than they incorporate since their triglyceride stores need to be mobi- 
lized. The epithelial cells at end of gestation, however, are rapidly 
synthesizing milk components as lactogenesis occurs, and rates of in- 
corporation catch up with rates of oxidation (figure I1I-6). 

Endocrine control of mammogenesis and lactogenesis, and their 
relationship to uterine development during pregnancy, have not been 
defined in pigs. Knight et al. (1977) described uterine, placental, 
and fetal development in gilts over the course of gestation. Uterine 
weight, placental weight, placental length, placental surface area, and 
allantoic and amniotic fluid volumes attained maximum development by 
day 60 or 70 of pregnancy. Increased estrogen production by the 






-62- 



fetoplacental unit between days 20 and 30 of pregnancy (Robertson and 
King, 1974; Knight et al. , 1977) may have stimulated mammogenesis in 
early pregnancy (figure III-3) , when the uterus and products of con- 
ceptus were developing rapidly. Between days 60 and 90 of pregnancy, 
when uterine and placental development essentially were complete, there 
was a 400-fold increase in estrogen output by the uterus and this con- 
tinued until term (Knight et al., 1977). This corresponded to the im- 
pressive increase in DFFT and DNA concentration observed between days 
75 and 90 (figure I1I-3) . Concentrations of progesterone were elevated 
in pregnant gilts at this time (Knight et al. , 1977), but decreasing 
progesterone and/or increasing estrogens by day 90 or 100 and are prob- 
ably related to increased mammary RNA concentrations and metabolic rate 
at the end of pregnancy. Thus, it is possible that suppression of 
lactogenesis by elevated concentrations of progesterone occurred until 
after day 90 of pregnancy. In fact, Martin et al. (1978) found a nega- 
tive correlation (r = -.80, P < .02) between concentration of plasma 
progesterone and concentration of lactose in mammary secretions of 
periparturient sows. After day 105 of pregnancy, decreasing progesterone 
and increasing estrogen concentrations (Robertson and King, 1974; 
Knight et al., 1977) were associated with the tremendous increase in 
metabolic activity of the mammary gland observed in this study. 



B. Ultrastructural Changes in Porcine Mammary Tissue 
During Lactogenesis 



Introduction 

As was previously stated (see Chapter III, Section A, Introduction), 
to understand the interaction between the gravid uterus and mammary 



■'*^— --r-;^-*->'i»M4l*.*^ . 



-63- 



function, one must understand the process of lactogenesis from a struc- 
tural as well as a biochemical standpoint. Previously, investigators 
have described the ultrastructural changes associated with lactogenesis 
in the rat (Chatterton et al., 1975), rabbit (Bousquet et al. , 1969), 
dog (Sinowatz et al., 1980), and cow (Feldman, 1961; Saake and Heald, 
1974). Unfortunately, little has been done to examine fine structural 
changes which occur in the mammary gland of the pig during lactogenesis. 
In his essay on comparative mammary fine structure. Wooding (1977) very 
briefly described the relative amounts of organelles present in mammary 
tissue from the pig on days 30 and 111 of pregnancy, or during lacta- 
tion. The current study was conducted to describe in greater detail 
the cytological changes which occur in mammary tissue from the gilt 
during periparturient period (from day 90 of pregnancy to day 4 post- 
partum) . 

Materials and Methods 

Tissue was obtained for ultrastructural analysis from the mammary 
biopsies performed in the previous study (Chapter III, Section A, 
Materials and Methods). Immediately after biopsy, tissue was cut into 
pieces of approximately 1 mm , and fixed for 2 hr in 2A glutaraldehyde 
buffered at pH 7.4 with .2 M Sym-Collidine (2,456-trimethyl pyridine; 
Polysciences, Inc. , Warrington, PA). After a 1 hr rinse in Sym-Collidine 
buffer, tissues were post-fixed in Sym-Collidine buffered osmium tetroxide 
(2%) at pH 7.4 for an addition hour . Tissues then were dehydrated in a 
graded series of ethanol solutions, equilibrated in acetone, and em- 
bedded in Spurr ' s Low Viscosity Embedding Medium (Polysciences, Inc.). 



-64- 



Subsequently 1 y thick sections of tissues were made with an LKB Ultra- 
tome III ultramicrotome, stained with toluidene blue, and observed with 
a Leitz light microscope, Ultrathin sections (60-100 nm) were then 
made, post-stained in uranyl-acetate and lead-citrate, and examined 
with a Hitachi HUUE electron microscope. 

From the previous study (Chapter III, Section A) it was determined 
that by day 90 of pregnancy near maximim concentrations of DNA had been 
attained in the porcine mammary gland (figure III-3) . Examination of 
RNA concentrations and the RNA to DNA ratio (figure III-4) indicated 
that after day 90 of pregnancy the mammary epithelial cells underwent 
differentiation as they acquired the capacity to synthesize and secrete 
milk. Therefore, tissues from days 90, 105, and 112 of pregnancy, the 
day of parturition, and day 4 of lactation were used for ultrastructural 
examinat ion . 

Results and Discussion 

Mammary tissue from a gilt on day 90 of pregnancy is shown in 
figure III-7. Two alveoli with a single layer of epithelial cells sur- 
rounding the lumina are visible in figure III-7,A. Also present are 
developing alveolar structures composed of solid balls of cells not 
having a distinct lumen visible demonstrating variation found in al- 
veolar lumen size on this day. Also visible are two blood vessels con- 
taining red blood cells and a lymphocyte (figure III-7,B). Preserva- 
tion of the normal biconcave structure of erythrocytes indicates no 
osmotic shock occurred during the fixation process. Secretions present 
in these tissues stained densely due to the high protein concentration 



r ---^j,^ 4>-3i ^..^■j.w*'^ -i.^' V >;Ti.«.w^ v.-.-i-- •■ — --W- ^- — i-- -,, ti-,— ^ fcr-j,..tf>wi^a-»5Jr3.-*> — ^^*~^:-^- • — -i— -;■-■ •--•uw-dt^ -^;"-.- -i-^ 



Figure III-7. Porcine mammary tissue on day 90 of pregnancy. 

A. General view. X320. B. A single epithelial 
cell. X9,000, C = capillary, L = alveolar lumen, 
N = nucleus, ER = smooth endoplasmic reticulum, 
ID = lipid droplet, G = golgi, M = mitochondrion. 



vr**!^*"— ''^■,- f '4*^— s*-**-"— ■ --"" — ■-^' .••-'*-•••»?•« '^li'* r.iJM'i'if'-* -";.= — . .^T,--|i»*ir . »* 7.*-]:-*ts iv,»-^'»fl 'f •■^(-->"- •• "j^' 



-66- 








-67- 



found in colostrum. In the basal portion of several cells one could 
observe areas occupied by small lipid droplets (figure III-7,A). A 
typical matranary epithelial cell on day 90 of pregnancy (figure III-7,B) 
had a large, irregularly shaped nucleus with one or two prominent 
nucleoli. A few lipid droplets were present in the basal portion of 
some cells (figure III-7,B), and numerous oval and rod-shaped mito- 
chondria (with visible cristae) were scattered throughout the cytoplasm 
(figure III-7,B). Some smooth endoplasmic reticulum was present in the 
basal cytoplasm of the cell, but very little rough endoplasmic reticulum 
existed and numerous free ribosomes could be observed. The golgi ap- 
paratus was a series of stacked plates, and was located in a paranuclear 
region of the cell. No golgi vacuoles or secretory vesicles were ap- 
parent, and the apical cell membrane contained a few short, club-shaped 
microvilli. These observations were similar to the description of 
midpregnancy mammary epithelium made by Hollmann (1974), and are con- 
sistent with the tissue-slice incubation study (Chapter III, Section A) 
which suggested that differentiation of epithelial cells of the mammary 
gland had not yet occurred. 

Thick sections of mammary tissue from day 105 of pregnancy (figure 
III-8,A) showed increased area occupied by alveolar lumina. At this 
time all alveoli in the field contained secretion. Other features were 
an increase in size of the cytoplasmic lipid droplets and an increased 
number of lipid droplets within the alveolar lumina (figure III-8,A). 
An electron micrograph of mammary epithelium on day 105 (figure III-8,B) 
suggested that the process of differentiation or lactogenesis had begxm. 
Secretory cells assumed a more cuboidal appearance as secretions began 
to accumulate within the lumina. Nuclei were more regular in shape 



Figure III-8. Porcine mammary tissue on day 105 of pregnancy. 

A. Several alveoli. X320. B. An epithelial and 
a myoepithelial cell. X4,600. L = alveolar lumen, 
MV = microvilli, G = golgi, LD = lipid droplet, 
N = nucleus, MC = myoepithelial cell. 



—_-^ii -.'';; ... 



-69- 



ai>^%7 ... 








*^.. 4a 



w »-J».i:;> ',.< r ;. | i;>^v.,iv.rTf»>*||U' 



l«— *S-i|*J«»V->- -UiiUV*H*ifc—fcHB^, 



• ••**1&*,'^T^*^P— W--fl*' 



-70- 



(figure III-8,B), and although the size of the lipid droplets had in- 
creased, they still occupied either a basal or paranuclear position. 
The golgi apparatus assxamed a more supranuclear position, but no secre- 
tory vesicles were evident. The cytoplasm was more electron-dense than 
on day 90, which at higher magnification (not shown) was shown to be 
due to an increased number of polysomes, but very little rough endo- 
plasmic reticulum was present. The apical cell membrane still contained 
only a few short microvilli (figure III-8,B). Changes in cellular 
organelles compared to day 90 tissue agreed with the increased RNA con- 
centration observed in the previous study (figure III-4) for this tissue. 
Increased area occupied by alveolar lumina suggested that in the pig, 
as in the dog (Sinowatz et al, , 1980), some secretion of colostrum takes 
place as early as ten days prepartum. 

By day 112 of pregnancy (figure III-9,A) the cytoplasmic area was 
dominated by lipid droplets and there was a slight increase in the num- 
ber of lipid droplets present in the secretion. However, the processes 
of synthesis and secretion were not yet fully coupled as the cells re- 
tained the majority of the lipid synthesized (figure HI-9,A). This 
also had been observed by Chatterton et al. (1975) in the prepartum rat 
mammary gland. On day 112 of porcine pregnancy the mammary epithelial 
cell nuclei, although still somewhat irregular in form, appeared to 
occupy a smaller portion of the cytoplasm than previously was observed 
(figure III-9,B). Numerous mitochondria were observed in all areas of 
the cell. While ntimerous polysomes remained throughout the cytoplasm, 
rough endoplasmic reticulum was observed in the basal aspects of some 
cells (figure III-9,B). A large portion of the cytoplasm was occupied 
by lipid droplets and some were in the process of being extruded from 



► — -^' 'tM« ■•'■■.I*,'" 



Figure III-9. Porcine mammary tissue on day 112 of pregnancy. 
A. A single alveolus. X400. B. An epithelial 
cell. X4,600. L = alveolar lumen, MV = micro- 
villi, M = mitochondria, N = nucleus, LD = lipid 
droplet, RER = rough endoplasmic reticulum. 






-72- 











-73- 



the cell. Although It was difficult to detect discrete areas of golgi 
apparatus, appearance of densely staining protein granules within the 
secretion suggested that the golgi was functional to some degree (figure 
III-9,B). Microvilli on the apical cell membrane had elongated by day 
112 compared to those in previously observed tissues. Mammary tissue 
of the gilt on day 112, only three days prior to parturition, apparently 
was similar in appearance to mammary tissue from several species in the 
"immediate prepartum period." Such tissue had been characterized as 
having little development of the rough endoplasmic reticulum, few 
secretory vesicles, and a large number of lipid droplets within the 
cell (Wooding, 1977). These ultrastructural observations also were 
consistent with changes in nucleic acids and metabolic activity. While 
increasing concentrations of RNA (figure III-4) on day 112 suggested 
that cells were acquiring the protein synthetic machinery necessary 
for lactation, metabolic activity estimates (figure III-6) indicated 
that these processes still were much less active than observed during 
lactation. 

Mammary biopsies on the day of parturition were performed approx- 
imately 6 hr after the last piglet was born, and after the litter had 
nursed the dam. Consequently, several micrographs of tissue from the 
day of parturition (figure III-IO,A) showed alveoli with relatively 
small lumina. Secretions within the majority of these lumina did not 
stain densely, but had the appearance of normal milk, suggesting that 
the colostrum had been removed. Nuclei of the epithelial cells were 
rounded, and lipid droplets were evident in the apex of most cells 
(figure III-10,A). At the electron microscope level (figure 111-10, B) 
one could observe milk of normal appearance within the lumina which 



Figure III-IO. Porcine mammary tissue on the day of parturition: 

Six hours post labor. A. A single alveolus. X400. 
B. A single secretory cell. X6,300. N = nucleus, 
L = alveolar lumen, G = golgi, MV = microvilli, 
LD = lipid droplet, RER = rough endoplasmic 
reticulum. 



t— ~ "SSIH^t^-^^ 



-75- 



y ^Sbc<^' 


















'•■'r.' ■*■ -.■^''- ■■■■ir>''"'"'5'''' 







- — • --"'•»w,fj^ ——•il »«■?-.,-,■_- ■ 



.«-».'. «JV*f ■!- -1 



-76- 



contained numerous lipid droplets and electron-dense protein granules. 
Nuclei were rounded in appearance, and the cytoplasm was rich in organel- 
les. Cells had considerable amounts of rough endoplasmic reticultim in 
the basal cytoplasm and adjacent to the nucleus. There were well-defined 
areas of expanded Golgi apparatus lateral and apical to the nucleus, 
and lipid droplets accumulated in the apical ends of the cells (figure 
III-10,B). Microvilli on the apical cell membrane had elongated com- 
pared to three days earlier, coincident with the process of active milk 
secretion. Mammary tissue from the day of parturition, with the rough 
endoplasmic reticulum-golgi arrangement, exhibited the distinct cellu- 
lar polarity characteristic of lactating mammary epithelial cells (Saake 
and Heald, 1974). Not surprisingly, these changes occurred during the 
period when the first significant increase in mammary llpogenesis was 
observed (figure III-6) . 

Mammary tissue on day +4 of lactation was characterized by alveoli 
with very large lumina (figure III-11,A), suggesting that the tissue 
was very active in milk synthesis and secretion. In fact, cells lining 
some alveoli were almost squamous in appearance. Most nuclei were 
rounded and had prominent nucleoli; and the apex of most cells con- 
tained numerous lipid droplets (figure III-11,A). Secretions within 
the lumina contained numerous densely staining granules and lipid 
droplets of various sizes. Ultrastructural examination of mammary 
secretory cells on day +4 of lactation showed a cytoplasm which was 
rich in organelles (figure III-11,B). Dilated cisternae of the rough 
endoplasmic reticulum were present throughout the cell, but were par- 
ticularly concentrated in the basal cytoplasm. The golgi apparatus was 
well developed with numerous dilated vacuoles and secretory vesicles 






Figure III-ll. Porcine mammary tissue on the fourth day of lacta- 
tion. A. Portions of two alveoli. X400. B. Two 
epithelial cells. X6,300. L = alveolar lumen, 
N = nucleus, G = golgi, RER = rough endoplasmic 
reticulum, SV = secretory vesicle, ID = lipid drop- 
let, V = microvesicle, MV = microvilli. 



-78- 





-79- 



present in the supranuclear region (figure III-11,B). In addition, 
numerous microvesicles were observed just inside the apical membrane 
of the cell. Secretory vesicles containing numerous condensed protein 
granules were being transported toward the lumina, and elongated micro- 
villi were present on the apices of all secretory cells. At this 
stage, numerous cells had lipid droplets which were in the process of 
being extruded into the lumen, suggesting that synthesis and secretion 
were fully coupled in typical cells in early lactation. Overall ap- 
pearance of cells on day +4 of lactation suggested that ultrastructural 
differentiation was complete. This was supported by the tremendous 
increase in rates of lipogenesis and substrate oxidation which were 
previously observed in the same tissue samples (figure III-6) . Results 
from the present study also agree with observations made by Wooding 
(1977). He observed a large increase in rough endoplasmic reticultmi 
and golgi vesicles, and a pronounced decrease in lipid droplets of 
lactating porcine mammary tissue compared to tissue from HI days of 
pregnancy. 

C. Summary 

Changes in mammary gland histology, dry weights, nucleic acid con- 
centrations, and in vitro rates of substrate oxidation and incorporation 
into lipid were measured in mammary biopsies of three gilts each on 
days 30, 45, 60, 75, 90, 105, and 112 of pregnancy, and days 1 and 4 
of lactation. Ultrastructural changes also were characterized between 
day 90 of pregnancy and the fourth day of lactation. Histological 
changes noted were progressive duct growth early in pregnancy followed 



-80- 



by rapid lobuloalveolar development between days 75 and 90 which was 
accompanied by a pronounced increase in DNA concentration (figure 
III-3) . Histology, and nucleic acid concentration changes, along with 
total DNA measurements (Hacker, 1970) and whole mount preparations 
(Turner, 1952) suggested that manmiary gland growth was essentially 
complete by day 90 of pregnancy in the gilt. Concentrations of RNA 
(figure III-4) , histology (figure III-2) , and metabolic activity 
measurements (figure III-6) , however, indicated that lactogenesis had 
not yet begun. Endocrine control of mammogenesis remains to be eluci- 
dated but estrogens certainly are implicated. On day 70 of pregnancy 
the porcine fetal-placental unit begins to produce significant amounts 
of estrogen (Knight et al. , 1977), and Hayden et al. (1979) showed 
that estrogen can induce the prolactin receptor in mammary glands of 
virgin rats. 

Rates of acetate and glucose oxidation by mammary tissue slices 
in vitro increased transiently during midpregnancy, then declined and 
remained low until initiation of lactogenesis (figure III-6) . Sub- 
strate incorporation into lipid increased slightly at midpregnancy 
and again at day 105, after which it increased markedly (figure III-6) . 

Preferential utilization of acetate, as opposed to glucose, by 
mammary tissue slices during the second half of gestation was con- 
sistent with the report by George et al. (1978) of a diabetogenic 
effect of pregnancy on maternal tissues. A greater utilization of 
acetate and glucose for oxidation rather than for lipogenesis in early 
and midpregnancy was abolished with the onset of lactogenesis and 
copious milk secretion (figure III-6) . This probably reflected two 
differences. The population of cells was different in early (adipocytes 



i!t.-m- .-<t-rf~ -^. »— r. 



-81- 



and epithelia) than in late pregnancy (mostly epithella) , and the 
functions of these cells were different. The primary function of the 
adipocytes and epithelial cells of early pregnancy were lipolysis and 
hyperplasia, respectively. In contrast, the function of the epithelial 
cells at the end of pregnancy was milk synthesis. 

Porcine mammary tissue on day 90 of pregnancy was composed of 
alveoli which contained negligible to moderate amounts of secretion. 
Epithelial cells of these alveoli were relatively undifferentiated 
(figure III-7) . While nximerous mitochondria were seen within each 
cell, lobulated nuclei occupied large portions of the cytoplasm, little 
or no rough endoplasmic reticulum or golgi apparatus was apparent, 
and little evidence of secretory activity was observed (figure III-7) . 
Ultrastructural features of the porcine mammary gland on day 90 were 
similar to those reported by Hollmann (1974) for midpregnant mouse 
mammary gland . 

Increased concentration of RNA (figure III-4) , histological 
evidence of colostrum (figure III-2) , the appearance and distribution 
of cellular organelles (figure III-8) , and the rate of lipogenesis from 
acetate (table III-3) suggested the process of differentiation had been 
initiated by day 105 of pregnancy in the gilt. A further increase in 
RNA (figure III-4) and metabolic activity (table III-3) , distension of 
the alveolar lumina (figure III-2) , and increased lipid and rough 
endoplasmic reticulum within the cells (figure III-9) suggested dif- 
ferentiation had progressed by day 112. On the day of parturition, 
with the initiation of milk removal, the process of milk synthesis was 
coupled with the process of secretion. Secretions within the alveolar 
lumina assumed the appearance of normal milk (as opposed to colostrum) 



-82- 



and the epithelia displayed the distinct cellular polarity character- 
istic of lactating mammary tissue (figure III-IO) . By day +4 of lac- 
tation, differentiation of epithelial cells appeared complete, with 
dilated rough endoplasmic reticulum and numerous secretory vesicles 
evident (figure III-ll). Elongated microvilli were present and numerous 
cells contained lipid droplets which were in the process of being ex- 
truded into the lumina. Ultrastructural observations on the day of 
parturition and the fourth day of lactation occurred at a time when 
pronounced increases in RNA concentrations and rates of fatty acid 
synthesis and substrate oxidation were observed (figures III-4, III-6) . 
Histology, cytology, metabolic activity, and nucleic acid measure- 
ments collectively indicated that lactogenesis in the pig occurred in 
two stages. Stage one occurred between days 90 and 105 of pregnancy, 
and stage two between day 112 of pregnancy and early lactation when the 
predominant feature was active milk secretion. Thus, the pig is 
similar to the rabbit (Strong and Dils , 1972; Mellenberger and Batiman, 
1974a, b) and cow (Mellenberger et al., 1973; Kinsella, 1975) in that 
lactogenesis stage one occurs several days prepartum. This does not 
agree with the report by Martin et al. (1978) that lactation in the sow 
is initiated within 24 hr of parturition. Since these investigators 
did not examine mammary tissue nor rates of milk synthesis, their 
definition of lactogenesis was actually lactogenesis stage two, or 
copious milk synthesis and secretion. Results from Martin et al. 
(1978) indicated that lactose in sow's milk was correlated negatively 
with concentrations of progesterone in maternal blood, and suggested 
that progesterone withdrawal controlled onset of lactogenesis stage 
two. 



-83- 



Evidence to date indicates that there is no placental lactogen in 
the rabbit or pig (Talamantes et al., 1980), and although secreted by- 
bovine placentae, placental lactogen may not reach the maternal circu- 
lation in the cow (Schellenberg and Friesen, 1981). It is interesting 
that lactogenesis is initiated well before parturition in each of these 
species. In rabbits, there is a marked increase in free prolactin 
receptors on day 20 of pregnancy (McNeilly and Friesen, 1977), coin- 
cident with lactogenesis stage one. This is in contrast to the rat 
which has a placental lactogen (Talamantes et al, , 1980) and a one 
stage lactogenesis occurring within 24 hr of parturition (Kuhn, 1968). 
Unoccupied prolactin receptors rise at parturition Instead of at mid- 
pregnancy in the rat (Holcomb et al., 1976; Hayden et al., 1979). 
However, nothing is known about the ontogeny of the prolactin re- 
ceptor in the mammary gland of the sow or cow, and a concensus needs 
to be reached about concentrations of placental lactogen in the circu- 
lation of the cow. 

Teleo logically, it may be advantageous to the fetus if placental 
lactogen inhibited lactogenesis prepartum, thereby promoting nutrient 
storage by other maternal tissues in anticipation of the need to 
mobilize nutrients for neonatal nutrition in the postpartum period. 
Clearly, the development of a unified hypothesis of the endocrine con- 
trol of lactogenesis depends upon clarification of the interaction 
between placental lactogen and the prolactin receptor in the cow. In 
the pig estrogen may be the hormone of conceptus origin which regu- 
lates prolactin receptors prepartum. However, this remains to be 
established. 



CHAPTER IV 

INFLUENCE OF CONCEPTUSES ON MAMMARY DEVELOPMENT 
AND LACTOGENESIS IN THE PIG 



A. Effect of Conceptus Number on Mammary Development in Gilts 



Introduction 



Results from the previous chapter supported the concept that the 
majority of mammary gland development occurred during pregnancy in the 
pig (Turner, 1952; Hacker, 1970). In addition, the large increase in 
mammary gland DNA concentration observed between days 75 and 90 of 
pregnancy (figure III-3) occurred just after completion of placental 
growth and at a time which corresponded to increased venous-arterial 
differences in estrogen concentration across the uterus (Knight et 
al., 1977). 

Previously, Desjardlns et al. (1968) showed that mammary gland 
growth in pseudopregnant rats was less than that of pregnant rats, and 
that removing fetal placentae caused mammary glands to regress. Wrenn 
et al. (1966) also had showed that fetal placentae were necessary for 
normal growth of mammary glands of rats, but attempts to induce normal 
mammary development in pseudopregnant animals by injections of pla- 
cental extracts were unsuccessful. This contrasted results of Leonard 
(1945) who believed that placentae could maintain the morphological 
integrity of the rat mammary gland during the second half of gestation 
and that they could do so in the absence of ovaries, pituitaries, and 



-84- I 






-85- 



fetuses. More recently, Nagasawa and Yanai (1971) found significant 
positive correlations between the number or weight of placentae and 
mammary gland DNA or RNA in mice. These data provide strong evidence 
for conceptus influence on mammary development in rats and mice, and 
the following data suggest the same is true for some domestic species, 
as well. 

Records of over one-thousand parturitions collected at the Uni- 
versity of Florida have demonstrated a positive relationship between 
calf birth weight and lactation performance in Holstein cows sug- 
gesting that fetal calf growth was associated with mammary gland growth 
(Thatcher et al., 1979). Bolander and Fellows (1976) reported that 
maternal concentrations of bovine placental lactogen, a hormone pro- 
duced by the fetal placenta, were related to subsequent milk yields in 
both dairy and beef cows. However, the concentrations reported by 
Bolander and Fellows (1976) have not been observed by others (Roy et 
al. , 1977; Hayden and Forsyth, 1979; and Schnellenberg and Friesen, 
1981). Hayden et al. (1979) and Butler et al. (1981) reported trends 
for goats and ewes with twins and triplets to have higher blood levels 
of lactogenic activity and better subsequent lactation records than 
animals giving birth to single offspring. It is interesting that 
evidence for a placental lactogen has been reported for all of these 
species (see review by Talamantes et al. , 1980). In constrast, no 
one has reported evidence for a placental lactogen in the pig. Several 
investigators have provided evidence that the pregnant porcine uterus 
is a rich source of hormones. Perry et al. (1973) and Fischer et al. 
(1981) showed the ability of the porcine conceptus to synthesize 
estrogens from steroid precursors, and Ainsworth and Ryan (1966) 



-86- 



demonstrated that porcine placenta could aromatize androgens to estro- 
gens. Knight et al. (1977) demonstrated large (greater than 1 ng/ml) 
differences in venous-arterial estrogen concentrations across the preg- 
nant uterus of the pig in early and late pregnancy, and Chew et al. 
(1979) and Stoner et al. (1980) have detected a positive relationship 
between estrone-sulfate and the number of conceptuses present in sows 
during gestation. 

Estrogen, progesterone, and prolactin are known to be mammogenic 
(Anderson, 1974). Since increased arterio-venous differences in estro- 
gen concentrations across the uterus were coincident with a period of 
rapid mammary development in pigs, an experiment was designed to deter- 
mine if treatments to manipulate conceptus number could alter the endo- 
crine status of gilts during pregnancy and thereby influence mammary 
gland development. 

Materials and Methods 

Thirteen crossbred gilts of similar weights, ages, and genetic 
background were utilized in three treatments which affected conceptus 
nximbers. Gilts rather than sows were used to avoid the effect of pre- 
vious pregnancy of lactation on mammary gland growth. Five gilts each 
received daily subcutaneous injections of 5 mg estradiol-valerate on 
days 11-15 of the third observed estrous cycle to induce pseudopreg- 
nancy. These formed a group of five gilts with zero fetuses. Four 
gilts were assigned to a second treatment in which a single oviduct 
was ligated to restrict the contribution of embryos to the uterus to 
only those from a single ovary. Five to ten days after the first 



,;»^.A;T>iij<^ — di--> ,-^- — li.- — -r^ji^r^n-* 



-87- 



observed estrous period each gilts was anesthetized with sodium thiamylal 
and anesthesia maintained with metofane. A mid-ventral laparotomy was 
performed and the ampulla of the left oviduct was doubled over and 
ligated with nxomber 1 milk suture. Each gilt in this group subsequently 
was bred to mature boars (at 12 and 24 hr after the onset of estrus) 
on the second estrus after surgery. An additional four gilts were bred 
by mature boars (twice) during the third observed heat period; these 
represented a group with a full complement of fetuses. 

Blood samples of approximately 20 ml were obtained from each gilt 
by puncture of the anterior vena cava every 10 days from day 10 through 
100 of pregnancy or pseudopregnancy. Additional samples also were ob- 
tained from pseudopregnant gilts on days 25 and 35 after onset of 
estrus. Samples were collected into heparlnized syringes and main- 
tained on ice until centrifuged at 8000 g for 15 min at 4 C. Plasma 
was collected and stored at -20 C until used for hormone analysis. 
Since the great majority of mammary gland development had occurred by 
day 110 of pregnancy (Chapter III; Hacker, 1970), day 110 was selected 
as the day of slaughter to allow maximum expression of conceptus ef- 
fects on mammogenesis without imposing upon stage 2 lactogenesis. ' 
All gilts were slaughtered on day 110 and a final bleeding, total j 
mastectomy, hysterectomy, and ovariectomy were performed on each gilt. i 
Immediately after each gilt was stunned and hung on the gambrel, blood 
was collected into an iced, heparlnized beaker, and plasma harvested i 
and stored as before. 

Mastectomy was Initiated by cutting through the skin around all 
mammary glands. Then, beginning in the inguinal region, glands were 
pulled away from the body by cutting through subcutaneous abdominal 






-88- 



fat down to the abdominal muscle layer, and then cutting in the cephalad 

direction, dorsal to the mammae. Care was taken to remove all mammary 

parenchymal tissue. In this manner, all mammary glands were removed 

from each gilt as a single piece of tissue. Skin was removed from the 

glands and they were dissected away from each other with care taken to 

section at the demarcation between glands. While it was realized that 

each porcine teat contains two streak canals and teat cisterns, each 

teat and its associated secretory tissue was treated as a single mammary { 

gland. Individual mammary glands then were trimmed of any muscle, ! 

i 
adipose, or connective tissue which did not contain mammary parenchyma. 

i 
Wet weights were recorded for each individual gland, and they were f 

I 
placed in labelled plastic bags, quickly frozen in liquid nitrogen, j 

and stored at -20 C until analyzed for nucleic acids. 

Soon after removing mammary glands, ovaries, oviducts, uterine 
horns, uterine body, and a portion of the cervix were removed from 
pregnant gilts and placed on ice. Ovaries were examined immediately 
and number of corpora lutea recorded. The remainder of the reproduc- 
tive tract was covered with ice and allowed to cool for 24 hr to permit 
easier dissection. To accomplish this, uterus was dissected free of 
oviductal and connective tissue and opened along the mesometrial border 
with care taken not to cut conceptuses. Each fetus and placenta was 
removed intact, transferred to a pan, and total number of fetuses re- 
corded. Fetuses were dissected away from placental membranes and 
individual fetus sex, crown-rump length, and wet weight was recorded. 
Placental wet weights and empty uterine weight also were recorded. 
Only number of corpora lutea was recorded for pseudopregnant gilts. 



i-,i|^~Jiy^^rt'^^.- &-■ -- -^T^'--,^. 



-89- 



When number of conceptuses finally was determined for each gilt, 
two of the animals with ligated oviducts had eight and ten fetuses 
compared to only seven fetuses each in two pregnant gilts which re- 
ceived no surgery. Since the purpose of the treatments was merely to 
create groups with different conceptus numbers, these four gilts were 
rearranged for all subsequent analyses resulting in five pseudopreg- 
nant gilts with zero conceptuses (group 1), four pregnant gilts with 
four-seven conceptuses (group 2) , and four pregnant gilts with eight-11 
conceptuses (group 3) . 

For nucleic acid measurements, individual mammary glands were 
thawed, representative subsamples removed from each gland, and minced 
into smaller pieces. A .5 g subsample of this material was then 
utilized for DFFT, DNA, and RNA determination as in Chapter III, Sec- 
tion A. This procedure was initiated by isolation of nucleic acids and 
phosphoproteins. The RNA and DNA were separated by the alkaline 
hydrolysis of RNA. The soluble RNA then was quantified by colorimetry 
using orcinol reagent. The insoluble DNA was then subjected to depuri- 
nization in 5% perchloric acid at 70 C for 15 min, and the resulting 
soluble purines quantified by absorption at 268 nm (Tucker, 1964). 
Concentrations of DFFT, DNA, and RNA then were multiplied by mammary 
gland wet weight to determine the total DFFT, DNA, and RNA in the 
gland, and nucleic acid concentrations on a DFFT basis were also de- 
termined. Plasma samples were analyzed by radioimmunoassay (RIA) for 
concentrations of progesterone, estrone, estradiol, and total estrogen- 
sulfate in this laboratory. In addition, an aliquot of each sample 
was sent to Dr. Robert Kraeling at the USDA Russell Research Center in 
Athens, Georgia, for measurement of porcine prolactin by RIA. 



«ii^'-* •-;. — — ...iH^i^*w^w^Wt>.J.«,-.- 



-90- 



Concentrations of progesterone in plasma were determined by RIA 
as described by Abraham et al. (1971) with an antibody (donated by J.L. 
Fleeger of Texas A and M University) which was highly specific (Eley 
et al., 1981). The assay was validated previously for porcine plasma 
in this laboratory (Knight et al. , 1977). During RIA of plasma samples 
in the current study, intra- and interassay coefficients of variation 
of ovariectomized-hysterectomized gilt plasma were 7.2 (n = 3)% and 
36.9 (n = 2)%, respectively. 

Estrone (E ) and estradiol (E ) were extracted from plasma with 
diethyl ether and isolated by column chromatography using Sephadex 
LH-20. Quantification of E, and E„ was accomplished by RIA as de- 
scribed by Chenault et al. (1975) and Eley et al. (1981) using an 
antibody donated by V.L. Estergreen (Washington State University). 
Validation of the assay in porcine plasma was reported by Knight et 
al. (1977), and intra- and interassay coefficients of variation for 
reference plasma run in the assays reported in the current experiment 
were 28.9 (n = 2.5) and 29.4 (n = 6)% for E^ , and 30.4 (n = 2.4) and 
39.9 (n = 6)%, respectively, for E„. 

To avoid the expensive and laborious column chromatography step 
used to isolate E and £„, a total estrogen-sulfate (E-SO.) procedure 
was developed and validated. Because of the anticipated range in con- 
centrations of estrone-sulfate between days 10 and 110 of pregnancy 
in gilts (see Robertson and King, 1974; Chew et al. , 1979; Hattersley 
et al., 1980; and Stoner et al. , 1980), the system was validated to use 
as little as 200 yl or as much as 2 ml plasma. The procedure was 
basically a modification of the estrone-sulfate assay reported by Eley 
et al. (1981), with exceptions being that different volumes of unknown 



. -^r^Liri*-- '*! ' 



-91- 

plasma were used, free estrogen was extracted with diethyl ether rather 
than benzene, and no column chromatography was utilized to separate 
unconjugated estrone from estradiol after cleavage of sulfated estro- 
gens. To insure that adequate recoveries could be achieved from 200 

3 
yl, 1 ml, or 2 ml of porcine plasma, 2000 cpm of estrone-6,7- H- 

sulfate, potassium salt (Sigma Chemical Co., St. Louis) was added to 
three replicates of each volume of ovariectomized-hysterectomized gilt 
plasma. After extraction of free estrogens with diethyl ether (five 
times the aqueous volume) , addition of acetate buffer (5 ml) and 12 hr 
incubation at 37 C with 20 U of sulfatase enzyme (isolated from Helix 
Pomatia, Sulfatase Type H-II crude; Sigma Chemical Co.) per tube, the 
hydrolyzed estrogen then was extracted with ether (2 extractions, at 
3 times the aqueous volume) . Recovery of radioactive hormone averaged 
85 . 3% and there was no apparent difference between the different volximes 
of plasma (range 84.1 to 86.4%). For all subsequent samples, internal 
recoveries were utilized in case of interassay or sample differences. 

To test for inhibition by plasma and accuracy of the assay 0, 100, 
150, 200, 400, 1000, 2000, 3000, 4000, 6000, 8000, or 10,000 pg of 
estrone-3-sulfate (estra-1 ,3,5 (10)-triene-17-one-3-sulfate) stabilized 
in potassium acetate was added to 200 yl or 2 ml volumes of ovariec- 
tomized-hysterectomized gilt plasma. This corresponded to 0, 70, 105, 
140, 280, 700, 1400, 2100, 2800, 4200, 5600, or 7000 pg of free E^ 
after correcting for the mass of the sulfate and the stabilizing com- 
pounds (product 70% free E as indicated by Sigma Chemical Company, 
and determined by molar extinction coefficient). After cleavage and 
extraction of the now unconjugated estrogen, the E^ RIA previously 
described was performed. Regression equations of added (X, in pg) 






-92- 



versus measured (Y, in pg) estrone were generated for the 200 yl and 
2 ml curves. There was no evidence of heterogeneity of regression. 
An overall pooled regression equation from both volumes of plasma was 
described by Y = 221 + .969X (R = .934), and the intraassay coeffi- 
cient of variation was 23.3%. For all the estrogen-sulf ate assays 
performed in this study the intra- and interassay coefficients of 
variation for a reference sample (28282 pg/ml) were 19.5 (n = 2.3) 
and 10.3 (n=6)%, respectively. 

A physiological validation of the E-SO, assay was demonstrated in 
that concentrations of the hormone increased in pregnant animals at 
the same stages of pregnancy as had been reported previously (Robertson 
and King, 1974; Hattersley et al., 1980). Interassay coefficients of 
variation for the steroid assays were high, but groups were balanced 
for assay differences since samples from each of the three treatment 
groups were represented in each assay. 

All data were analyzed by least squares regression according to 
the Statistical Analysis System (Barr et al., 1976). One-way analysis 
of variance was performed to examine the effect of treatment group on 
number of corpora lutea, number of conceptuses, sex ratio, mean and 
total fetal and placental weights, crown-rump lengths, and uterine 
weight. An analysis of variance of hormone concentrations was per- 
formed in which the variation due to group, gilt (group), day, and 
the group by day interaction was determined. Since gilts were nested 
in group, the gilt (group) mean square was used to test the main effect 
of group. 

Mammary gland variables for each gilt also were subjected to an 
analysis in which the effect of treatment group was examined by 






-93- 



performing two orthogonal comparisons of the three treatment groups. 
Since one objective of the study was to relate plasma hormone concen- 
trations to conceptus development or to mammary development, an 
average concentration of each of the hormones was calculated for each 
gilt by taking the mean of the 11 samples between day 10 and 110 of 
pregnancy or pseudopregnancy. Since the day 110 plasma sample was 
taken at slaughter, a large increase in prolactin concentration observed 
in several gilts was believed to be stress-related. Thus, only the 
samples from day 10-100 were utilized to calculate the prolactin mean 
for each gilt. Subsequently among-animal correlations were determined 
between the mean hormone concentrations in each gilt and each single 
measure of mammary, conceptus, uterine, or ovarian development. 
Finally, within-treatment correlations for mean hormone concentrations 
and mammary gland variables were determined, and after a test to deter- 
mine if they came from a common rho (population correlation coefficient), 
were pooled according to Snedecor and Cochran (1969). 

Results and Discussion 

Least squares means of uterine variables and number of corpora 
lutea are presented in table IV-1 for group 1 gilts with zero concep- 
tuses, group 2 gilts with four-seven conceptuses, and group 3 gilts 
with eight-11 conceptuses. The number of corpora lutea averaged 8.8, 
11.7, and 13.3 in groups 1, 2, and 3, respectively; but there was no 
significant effect of group (table IV-2) . Since grouping of animals 
was based upon the number of fetuses, the average was 0, 5.75, and 
9.25 in groups 1, 2, and 3, respectively; and were different (P < .001, 



rrt '—r>-'-'l T"T ■- — =" *.->-^)'* 



-94- 



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-95- 



table IV-2) . While it was hoped that group 3 animals would average 
well over ten pigs per litter, the experiment was initiated in July 
and perhaps heat stress affected embryonic survival. On the other 
hand, the 9.25 pigs per litter observed in group 3 gilts was similar 
to the 9.9 pigs per gilt observed by Knight et al. (1977) in the same 
herd. It also is possible that litter sizes were not greater because 
gilts rather than sows were used. Nevertheless, clear differences re- 
mained between treatment groups in number of conceptus units per gilt 
which allowed a test of the original hypotheses. Not surprisingly, 
there was no difference in sex ratio between groups 2 and 3 (tables 
IV-1, IV-2), which averaged 53.5% males. Mean fetal weight averaged 
1102 g in group 2 gilts and 1117 g in group 3 gilts (table IV-1) which 
were not different (table IV-2). This suggested that adequate uterine 
surface area was available to each embryo and fetus in group 3 gilts, 
that each could develop to its potential; assuming other factors such 
as nutrients were not limiting. A comparison of total fetal weight 
therefore reflected the difference observed in fetal numbers so that 
group 3 gilts averaged 10,256 g of fetus compared to only 6,173 g in 
group 2 gilts (P < .01, tables IV-1, IV-2). 

Mean placental weight averaged 217 g in group 2 fetuses and 198 g 
in group 3, which were not different (tables IV-1, IV-2), and total 
placental weights again reflected the difference in conceptus numbers 
as group 3 gilts had approximately 50% more total placenta (P < .10) 
than gilts in group 2 (tables IV-1, IV-2). Mean fetal crown-rximp length 
averaged 25.4 cm in group 2 gilts and 26.6 cm in group 3 animals and 
also were not different (tables IV-1, IV-2). No differences were ob- 
served in empty uterine weights (tables IV-1, IV-2). Observed values 



-96- 



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-97- 



for mean fetal weight, crown-rump length, and empty uterine weight were 
similar to those observed by Knight et al. (1977) on day 100 of preg- 
nancy in gilts from the same herd, and mean placental weights observed 
appeared to be slightly less than (approximately 15%) those observed 
by Knight et al. (1977) . 

Treatment group, individual gilts within group, day of pregnancy, 
and group by day interaction were investigated for their effects on 
concentrations of hormones in plasma of the gilts in the present study 
(table IV-3) . Treatment group significantly affected concentrations 

of E-SO, and E, , but not E^, progesterone, or prolactin (table IV-3). 

4 1 / 

Concentrations of E-SO,, E , E^, and progesterone differed among gilts 
but not prolactin (table IV-3). Concentrations of all hormones changed 
throughout the course of pregnancy, and a significant group by day 
interaction was detected for E-SO,, E^^, and Y.^, but not for progesterone 
or prolactin (table IV-3). Consequently, least squares day means were 
plotted for each group for E-SO,, E , and E^j but overall least squares 
day means were plotted for progesterone and prolactin (figures IV-1 
through IV-5) . Estrogen-sulfate concentrations (figure IV-1) were 
less than 2 ng per ml on day 10 and day 20 of pregnancy in groups 1, 
2, and 3 (figure IV-1). By day 30, however, conjugated estrogen had 
increased to 7.7 ng/ml in group 3 gilts and to 7.4 ng/ml in group 2 
gilts, but to only 1.7 ng/ml in pseudopregnant gilts bearing no con- 
ceptuses. An increase in sulfated estrogens on day 30 of pregnancy in 
pigs previously had been reported by Robertson and King (1974) , 
Hattersley et al. (1980), and Stoner et al. (1980) and is believed 
to be due to estrogen synthesis by the porcine conceptus (Perry et al. , 
1973; Ainsworth and Ryan, 1966; and Fisher et al. , 1981). Concentrations 



-98- 






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-101- 



of E-SO observed in groups 2 and 3 are approximately 60% greater than 
4 

the estrone-sulfate concentrations reported on day 30 by Robertson and 
King (1974), or Chew et al. (1979), and are 40% higher than those re- 
ported by Stoner et al. (1980). Two explanations for this discrepancy 
are plausible. The assay utilized in the current study did not dif- 
ferentiate between E -sulfates and E -sulfates and therefore would 
measure them both. Secondly, the assay utilized in the current experi- 
ment used a sulfatase preparation which also has glucuronidase activity 
and estrogen-3-glucuronides also would be measured in this system 
which could have inflated the E-SO estimate. In contrast, assays of 
Robertson and King (1974) and Chew et al. (1979) utilized a solvolysis 
procedure which presumably did not hydrolyze the estrogen-glucuronides. 
Concentrations of estrone-sulfate reported by Stoner et al. (1980) on 
day 30 of pregnancy (X = 5.2 ng/ml) were more similar to those reported 
in the current study, and not surprisingly, utilized the same sulfa- 
tase enzyme preparation. Concentrations of the conjugated estrogen in 
pseudopregnant animals also were elevated on days 20, 25, and 30 of 
pseudopregnancy compared to days 10 or 40 (figure IV-1) . This could 
have been due to formation of estrogen-conjugates as a result of general 
metabolism of the injected estradiol-valerate used to induce pseudo- 
pregnancy (on day 11-15), or perhaps were due to induction of sulfo- 
transferase activity within the endometrium, also as a consequence of 
the estradiol injections. 

Concentrations of conjugated estrogens in plasma declined in all 
gilts by day 40 of pregnancy or pseudopregnancy, and remained below 
1 ng/ml until day 80 of pregnancy (figure IV-1). At this time, con- 
centrations began to increase and continued to increase until day 110 



-102- 



of pregnancy at the time gilts were slaughtered (figure IV-1) . Concen- 
trations of E-SO, were related to the number of conceptus units in this 
4 

latter phase. Mean E-SO concentration in group 3 gilts was greater 
than mean concentration in group 2 gilts on each subsequent day observed 
(figure IV-1). Gilts in group 3 averaged 38 ng E-SO per ml compared to 
25 ng per ml in group 2 animals (figure IV-1) on day 110. Conjugated 
estrogens remained well below 1 ng/ml in pseudopregnant gilts until they 
increased very slightly on day 110 of pseudopregnancy (figure IV-1). 
This increase was due primarily to an elevated hormone concentration in 
one pseudopregnant gilt which at slaughter was determined to have a 
cystic follicle. 

The only report of concentrations of estrone-sulfate in pig plasma 
during late pregnancy was that of Robertson and King (1974) and was 
measured in a single animal. Because of the difference in conjugated 
estrogen assays mentioned previously, it is difficult to compare con- 
centrations directly. While Robertson and King (1974) observed a concen- 
tration of 3 ng per ml just prior to parturition, gilts in the present 
study exhibited concentrations 8-10 times higher (figure IV-1) . The 
difference between observations in the current study and those of 
Robertson and King (1974) were much greater near parturition than on 
day 30, and possibly suggest increased concentrations of estradiol-sulfate 
or the estrogen-glucuronides at this time. It is also possible that the 
concentrations in the gilt used by Robertson and King (1974) were un- 
usually low. Present study detected appreciable variability among gilts 

in their concentration of E-SO,. Nonetheless, the time of increased 

4 

E-SO, concentration noted in the current investigation was similar to 
4 

that reported by Robertson and King (1974). 



-103- 



Plasma concentrations of E for gilts in groups 1, 2, and 3 are 
depicted in figure IV-2 and indicate a profile similar to that observed 
for E-SO , except that no distinct increase In E occurred at day 30 in 
pregnant animals (figures IV-l, IV-2). Concentrations of estrone were 
low, and variable in all treatment groups from day 10 through day 40 of 
pregnancy (figure IV-2) . While mean concentrations of E in pregnant 
gilts on days 30 and 40 of pregnancy were higher than gilts in group 1, 
differences were not significant because of high variability. Subse- 
quently, E concentrations were uniformly low (< 80 pg/ml) in all 3 groups 
on days 50 and 60 of pregnancy (figure IV-2). Although low, concentra- 
tions for all three treatment groups were twice as high on day 70 as day 
60, and by day 80, free E, was higher in pregnant gilts (groups 2 and 3) 
than in those gilts with zero fetuses (figure IV-2). Knight et al. (1977) 
had observed previously a 15-fold increase in uterine vein-radial vein 
estrone concentration between days 60 and 70, and a further 7-fold in- 
crease between days 70 and 80. On day 90 of gestation, E concentrations 
in the current study averaged 1276 pg/ml in group 3, 404 pg/ml in group 
2, and 82 pg/ml in group 1 (figure IV-2) suggesting a relationship between 
conceptus numbers and concentrations of free estrogen. This trend con- 
tinued through day 110 as group 3 gilts averaged 3443 pg/ml, group 2 
gilts averaged 2767 pg/ml, and pseudopregnant gilts only 214 pg/ml (which 
was inflated due to one gilt with a cystic follicle). The estrone pro- 
file observed in pregnant gilts in this experiment was similar to those 
reported by Robertson and King (1974) and Knight et al. (1977). Con- 
centrations were quite low in early pregnancy, and the onset of the 
prepartum increase in E occurred at day 70 or 90 of pregnancy (figure 
IV-2). Average concentration of E in the pregnant animals in the current 



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-106- 



study was 3105 pg/ml on day 110 which was similar to the 2500 pg/ml 
observed just prior to parturition by Robertson and King (1974), or the 
3400 pg/ml observed in the uterine vein of gilts on day 100 of pregnancy 
(Knight et al. , 1977) . 

Variation in concentrations of E„ in pregnant and pseudopregnant 
gilts was greater than for any of the other estrogens (see SEM bars, 
figure IV-3) . While all gilts had relatively low concentrations of E^ 
on day 10, it was elevated temporarily (259 pg/ml) in pseudopregnant 
gilts on day 20, as a result of the estradiol-valerate injections used 
(figure IV-3) to induce pseudopregnancy. Through day 80, concentrations 
of E„ in the two pregnant groups of gilts remained below 80 pg/ml except 
for a dramatic increase in group 2 gilts on days 40 and 50 of pregnancy 
(figure IV-3) . This was due to a tremendously high value in one gilt 
(but not the same gilt) on each of the days, without which group 2 means 
would be 117 and 28 pg/ml on days 50 and 50, respectively. On days 60 
and 70, average concentration of E was less than 80 pg/ml in each group 
(figure IV-3) , but subsequently increased in pregnant gilts (groups 2 
and 3) and attained a concentration of 520 pg/ml on day 110 of pregnancy. 
The unusually high E„ concentration in group 2 gilts on day 90 may be 
accounted for by one extremely high value, without which the mean would 
be 77 pg/ml for group 2. This was comparable to the pseudopregnant gilts 
(figure IV-3). Based on the standard error, and the concentrations of 
estradiol on day 110, it appeared that estradiol was not related to litter 
size in the pregnant gilts, but estradiol concentrations clearly were 
higher in pregnant than in pseudopregnant animals (figure IV-3) . Con- 
centrations of E in all pseudopregnant gilts (including the cystic 
animal) increased slightly on day 110, perhaps suggesting some follicular 
activity (figure IV-3) . 





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ciw/gd] "ini<]yyi53 



-109- 



Concentrations of E measured in the current study were almost 10- 
fold greater than those observed in the uterine vein of pregnant gilts 
by Knight et al. (1977), and were much greater on day 110 (520 pg/ml) , 
than the 45 pg/ml previously found by Molokwu and Wagner (1973) , but dif- 
ferent from those reported by Robertson and King (1974). Differences 
between these studies were that Knight et al. (1977) sampled from the 
uterine vein of anesthetized gilts, and Molokwu and Wagner (1973) sampled 
from catheters placed in the anterior vena cava, while blood was obtained 
by venipuncture (of the vena cava) in the current study and by Robertson 
and King (1974). However, stress of venipuncture seems an unlikely ex- 
planation. Unfortunately, the specificities of the antibodies utilized 
cannot account for these differences either since the antibody used by 
Knight et al. (1977) was the same as that utilized in the current study. 

Overall day means for progesterone concentrations were plotted 
(figure IV-4) since the mean concentration of progesterone was not dif- 
ferent among groups, and there was not a group by day interaction (table 
IV-3) . Concentrations of progesterone in all 13 gilts averaged 24.6 
ng/ml on day 10 of pregnancy or pseudopregnancy (figure IV-4) , and de- 
creased in the subsequent 10 days to 14.2 ng/ml. Very similar concentra- 
tions and changes have been reported by Robertson and King (1974). 
Between days 20 and 100 of pregnancy or pseudopregnancy concentrations 
of progesterone averaged approximately 16 ng/ml, with transient increases 
observed on days 40, 70, and 110 (figure IV-4). This average progestin 
concentration was similar to values reported previously (Killian et al. , 
1973; Robertson and King, 1974). It was not known, whether there was any 
physiological significance to increased progesterone on days 40, 70, and 
110 of pregnancy, as this was not observed by Robertson and King (1974). 





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[ iN/aN : 3Nny3i533nyd 



-112- 



In fact, Robertson and King (1974) reported a decrease in concentration 
of progesterone between days 100 and 110, and Knight et al. (1977) noted 
a decrease in concentration of progestins between days 80 and 100 of 
pr-egnancy. In contrast, no decrease was observed before day 110 by 
Killian et al. (1973) or Martin et al. (1978), and Shearer et al. (1972) 
noted an increase between days 93 and 112 of pregnancy, similar to the 
profile observed in the current experiment. 

Concentrations of prolactin did not differ for the different treat- 
ment groups (table IV-3) , and were thus plotted as least squares day 
means (figure IV-5) . Concentrations of prolactin averaged 30.7 ng/ml on 
day 10 of pregnancy or pseudopregnancy (figure IV-5) but declined to 
14.0 ng/ml on day 20. Whether this concentration on day 10 was physio- 
logically important is questionable since the concentration was very 
high in only 3 of 13 gilts, and was not observed by Dusza and Krzymowska 
(1981). Mean concentrations of prolactin were constant (between 13.7 
and 18.3 ng/ml) between days 20 and 100 of pregnancy or pseudopregnancy 
(figure IV-5). This was similar to, but higher than, concentrations 
reported by Dusza and Krzymowska (1981) which averaged approximately 
7 ng/ml between days 10 and 68 of pregnancy. On day 110 of pregnancy 
concentrations of prolactin averaged 57.3 ng/ml compared to the 10 ng/ml 
observed in sows on day 110 by Dusza and Krzymowska (1981). These latter 
workers also showed that the prepartum increase in plasma prolactin did 
not begin until two days prepartiim. Reason for the greater concentration 
of prolactin observed on day 110 in the present study was undoubtedly 
due to the stress of slaughter, since Threlfall et al. (1974) measured 
concentrations of prolactin greater than 100 ng/ml in slaughtered sows 
at mid-gestation, when basal concentrations of prolactin were low (Dusza 



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■115- 



and Krzymowska, 1981; present data). Results from the present experi- 
ment indicated prolactin in maternal plasma was similar in pregnant and 
pseudopregnant gilts, which suggested little, if any, contribution from 
the placenta. 

Mean concentration of each of the hormones was calculated for each 
gilt and correlated with the number of corpora lutea, ntimber of fetuses, 
total fetal weight, total placental weight, or the empty uterine weight 
for each gilt (table IV-4) . Because the elevated prolactin concentration 
observed on day 110 was probably an artifact because of sample collection 
at time of slaughter rather than a physiological phenomenon, only values 
from days 10 through 100 were utilized to calculate a prolactin mean for 
each gilt. 

Results of the among animal correlations indicated there was a 
positive association (P < .05) between estrogen-sulfate concentrations 
and total fetal weights, and highly significant positive associations 
between estrogen-sulfate and number of fetuses, total placental weights, 
and empty uterine weights (table IV-4). However, estrogen-sulfate was 
not correlated with number of corpora lutea. Like E-SO, , E, was posi- 
tively correlated with the number of fetuses (P < .01) and empty uterine 
weight (P < .05) , but not with ntmiber of corpora lutea, total fetal 
weights, or total placental weights (table IV-4). Correlations of estro- 
gens with uterine variables was not surprising since a positive rela- 
tionship had been demonstrated between number of fetuses and estrogen- 
sulfate or E concentrations during pregnancy (figures IV-1, IV-2) . 
Concentrations of estradiol and prolactin were not significantly asso- 
ciated with number of corpora lutea or any of the uterine variables 
(table IV-4) , possibly indicating that estradiol was primarily of ovarian 



-^■^ir=- v*-^'i"iKn-a7«-T'*'^"»** 



-116- 



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-117- 



origin, and that prolactin was primarily from the maternal pituitary 
and not affected by secretions of the conceptus. However, the clear 
increase in estradiol of pregnant but not pseudopregnant gilts after 
day 70 of pregnancy suggested that there was f etal-placental production 
of estradiol, as observed by Knight et al. (1977). Therefore, estradiol 
in maternal plasma during pregnancy was probably of conceptus and 
ovarian origin. Progesterone was highly correlated (P < .01) with number 
of corpora lutea (table IV-4) , as was expected, but was not associated 
with any of the uterine variables. This supports the hypothesis that 
there is little, if any, placental contribution to maternal progesterone 
in the pig. 

Among animal correlations between the different hormones measured 
indicated that E-SO, was positively associated with E (P < .001) and 
with E„ (P < .05), but not with progesterone or prolactin (Appendix I). 
Similarly, E^ was correlated with E„ (P < .01). That estrogens were 
associated was expected since all estrogens were higher in pregnant gilts 
than in pseudopregnant animals. There was not a significant association 
between estrogen and progesterone, or progesterone and prolactin (Appen- 
dix I). Thus, there was no evidence that these steroids influenced 
progesterone or prolactin in the pig. 

Least squares means of various measures of mammary development in 
gilts from groups I, 2, and 3 are in table IV-5, and an analysis of 
variance in table IV-6 . Average number of mammary glands in each gilt, 
which could influence total mammary weight, was 13.4, 14.0, and 13.3 in 
groups 1, 2, and 3, respectively (table IV-5), and was not different 
between groups (table IV-6) . Total mammary gland wet weight averaged 
712, 2460, and 2330 g in groups 1, 1, and 3, respectively (table IV-5). 



-118- 



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-120- 



Analysis of variance indicated that group 1 had significantly less 
mammary tissue than groups 2 and 3, but that groups 2 and 3 were not 
different (table IV-6) . While there was a trend for DNA per g wet mammary 
tissue to increase with increasing litter size (table IV-5) , this trend 
was not significant (table IV-6) . Concentration of DNA on a per g dry 
fat-free tissue basis averaged 11,600 and 10,697 g in groups 2 and 3, 
respectively, versus only 8,352 g in group 1 (P < .01, table IV-6). Con- 
centration of DNA on a dry tissue basis did not differ between groups 2 
and 3 (table IV-6). Similarly, total DNA averaged 585, 2782, and 2479 
mg in groups 1, 2, and 3, respectively. Pseudopregnant gilts had only 
22% of the DNA as mammary glands of pregnant animals (P < .001), but with 
total DNA in pregnant gilts was not related to fetal numbers (table 
IV-6) . 

Mammary gland DNA had not been examined previously in pseudopreg- 
nant gilts. Wrenn et al. (1966) indicated that mammary glands of pseudo- 
pregnant rats had comparable development to those of pregnant rats on 
day 10 of pregnancy. Desjardinset al. (1968) reported that mammary 
glands of pseudopregnant rats had 70% of the DNA found in pregnant rat 
mammary glands on day 12 of pregnancy. However, by day 21 of pregnancy 
or pseudopregnancy, mammary glands of pseudopregnant rats (with or 
without deciduomas) had only 39% of the DNA as their pregnant counter- 
parts. This led researchers to hypothesize that maternal hormones were 
primarily responsible for mammary gland development in the first half 
of pregnancy in the rat, but that hormones from the fetal placenta 
tissues become the primary limiting factor for mammary development in 
the second half of pregnancy (Desjardinset al. , 1968). 



-121- 



Total DNA observed in mammary glands of pregnant gilts in the cur- 
rent study was only one-third of that measured in gilts by Hacker and 
Hill (1972), and DNA concentrations were only one-third of those measured 
in mammary tissue of gilts on day 112 of pregnancy in Chapter III. While 
not certain, this difference probably was due to a difference in the 
depurinization procedure which was necessitated because of equipment 
failure. In the nucleic acid analysis of Chapter III, an instrument 
sterilizer was utilized for the 70 C incubation. As the sterilizer was 
no longer operating at the time of the current investigation, a drying 
oven was utilized. The decreased efficiency of convective versus con- 
ductive heat transfer may have been responsible for the lower DNA values 
reported. All nucleic acid determinations in the current study were 
performed using an identical procedure, and mammary tissue from all 3 
groups was analyzed in each assay, so differences in DNA concentrations 
between treatment groups still would have been detected. 

Failure to detect a difference in mammary DNA between groups 2 and 
3 suggests that regulation of mammary development was different in pigs 
than in mice, in which mammary gland DNA was linearly related to the 
number of conceptuses (Nagasawa and Yanai, 1971). The relationship 

between conceptus number and total mammary gland DNA in gilts was best 

2 
described by the quadratic equation Y = 577.9777 + 583.4840X - 38.0241X 

2 
(P < .02, R = .78), where Y = total mammary gland DNA in mg and X = 

conceptus number. However, if the pseudopregnant gilts were omitted 
from the analysis then the quadratic relationship was no longer signifi- 
cant. Among pregnant gilts the relationship between conceptus numbers 
and total mammary DNA was best described by the cubic equation Y = 
-19820.3861 + 9813.5688X - 1334.3190X^ + 57.1957X-^ (P < .01, R^ = .86) 
where Y = total mammary DNA (mg) and X = conceptus number. Since data 



-122- 



were not gathered in gilts with less than four, but more than one, con- 
ceptus we do not know how many conceptuses are required for full mammo- 
genic response. 

Concentrations of RNA per g wet mammary tissue or per g dry fat-free 
tissue indicated that pseudopregnant gilt mammary glands had only 52-64% 
of that found in glands from pregnant gilts (tables IV-5 , IV-6). However, 
mammary RNA was not greater with greater conceptus numbers (groups 2 
versus 3). Because of differences in mammary wet weights, differences 
in total RNA were magnified between pregnant and pseudopregnant mammary 
glands, so that pseudopregnant glands had only 23-24% of the RNA found 
in glands from pregnant gilts (tables IV-5, IV-6), but groups 2 and 3 
were not different (table IV-6) . 

Ratio of RNA to DNA averaged 3.50, 3.93, and 4.36 in groups 1, 2, 
and 3, respectively (table IV-5); although group 1 was less than groups 
2 and 3 (P < . 10) , group 2 RNA:DNA was not significantly less than that 
of group 3 (table IV-6) . 

Concentrations of RNA observed in gilts in groups 2 and 3 were about 
10% less than those reported in Chapter III for gilts from day 112 of 
pregnancy. Total RNA found in mammary glands from the eight pregnant 
gilts of the current study was only 62.4% of that reported by Hacker and 
Hill (1972) for 2 animals. Animal variation and assays utilized may 
account for differences between studies. Ratios of RNA to DNA observed 
in group 3 gilts were approximately twice those reported by Hacker and 
Hill (1972) , and about 3-fold higher than those observed in gilts on day 
112 (in Chapter III) . This difference can be accounted for solely by 
the decreased estimate of DNA in the present study. 

Interestingly, mammary glands from pseudopregnant rats on day 21 
had 24% of total RNA of their pregnant counterparts (Desjardins et al.. 



■123- 



1968) , while pseudopregnant gilt mammary glands in this study also had 23 
to 24% of the total REA as mammary glands from pregnant gilts. As was 
true for DNA, mammary RNA was no greater in gilts with 8 to 11 conceptuses 
than in gilts with 4 to 7 conceptuses. This suggested that regulation of 
mammary gland RNA in gilts was different from mice in which a linear 
relationship existed between conceptus numbers and mammary gland RNA 
(Nagasawa and Yanai, 1971). 

To complement nucleic acid observations, maimnary tissue histology 
was compared in glands from pregnant and pseudopregnant gilts (figure 
IV-6). In both tissues, several lobules of mammary parenchyma could be 
observed which were surrounded by connective tissue bands and areas of 
white adipocytes (figure IV-6). However, lobules of pregnant gilts were 
further developed than those of pseudopregnant mammary tissue, reflecting 
the greater amount of DNA (table IV-5) . In addition, tissue from pseudo- 
pregnant gilts had more adipose and connective tissue, as the gland had 
not grown all the way into the mammary fat pad (figure IV-6). Secretory 
tissue present in the pseudopregnant animal appeared to be normal other 
than the fact that there were more large and small ducts present (figure 
IV-6) . Appearance of greater lipid and total secretion in the alveoli 
of pregnant animals reflected the difference in RNA to DNA ratio which 
had been detected (figure IV-6, table IV-5). 

Results suggest that regulation of mammary development in pseudopreg- 
nant rats and pigs was similar and therefore, maternal regulation of mammo- 
genesis during early pregnancy in these two species may have been similar. 
It also was clear that conceptus presence was required for normal mammary 
development in the pig, but they were unlike mice in which a significant 
linear relationship existed between conceptus numbers and mammary 
development. 



Figure IV-6. Histological photomicrographs of porcine mammary 
tissue on day 110 of pregnancy (A) or pseudopreg- 
nancy (B) . Magnification X16. 



-125- 









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-126- 



Placental lactogen has been identified in rats and mice, but not in 
pigs (Talamantes et al. , 1980). However, in pigs there are clear differ- \ 

ences in conjugated estrogen and free estrone with different numbers of j 

conceptuses. Perhaps, estrogen is the conceptus mammogen in pigs. 

To test the hypothesis that hormones affected by conceptus number f 

could Influence mammary development (and to determine if hormones not 

affected by conceptus number could alter mammogenesis) , among animal cor- j 

t 
relations between mean hormonal concentrations and various measures of i 

mammary development in each animal were determined (table IV-7) . Estrogen- |, 
sulfate was correlated significantly with mammary wet weight, DNA concen- 
tration, total DNA, RNA concentration, and total RNA, but not RNA to DNA | 
ratio (table IV-7) . Correlation coefficients for E and each of the 

mammary variables all were positive, but only those for DNA and RNA con- [ 

centrations were significant (P < .05). Concentrations of estradiol 
were associated positively (P < .01) with DNA concentrations, but not 
to other measures of mammary development (table IV-7) . Concentrations 
of progesterone apparently had a very minor (although negative) effect j 

on mammary variables (table IV-7). Although mean prolactin concentra- 
tion was not different between the treatment groups (table IV-3) pro- 
lactin was positively correlated (P < .10) with mammary wet weight, but 
not with other measures of mammary development (table IV-7). 

The positive associations of E-SO, and E, with all measures of mammary 
development was not surprising since estrogens are mammogenic (Anderson, 
1974) and concentrations were related to fetal numbers. Correlation coef- 
ficient of .721 indicated that 52% of the variation in total mammary DNA 
was accounted for by variability in conjugated estrogens. The mechanism 
of the estrogen effect on the mammary epithelium is not known. However, 
it would be interesting to know if estrogen can induce prolactin receptors 



-127- 





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-128- 



in the porcine mammary gland, as it can in the rat (Hayden et al. , 1979). 
The possibility that estrogen stimulated prolactin secretion in pregnant 
pigs, as it does in rats (Neill, 1974) and sheep (Vician et al. , 1979), was 
excluded since prolactin concentrations of gilts in group 2 and 3 were not 
greater than those measured in pseudopregnant animals at the end of gesta- 
tion. The fact that prolactin was positively associated with wet mammary 
weight may be due to its effect on water movement into the gland. Analy- 
sis of prolactin data (table IV-3) did not demonstrate a gilt effect. 
However, when the 110 (stressed) sample was omitted to calculate gilt 
means for correlation purposes, these means ranged from 11 to 30 ng per ml. 

From measurements of DNA made (Chapter III) it was apparent that 
maximum concentrations were attained by day 90 of pregnancy. Total mastec- 
tomies were not performed in that study, but from DNA concentration changes 
(Chapter III) and total mastectomy data of Hacker and Hill (1970), it was 
evident that at least 77% of mammary development had occurred by day 90 
of pregnancy. In addition, a majority of mammary growth during pregnancy 
occurred between days 75 and 90 (figure III-3) . For this reason, mean 
concentrations of hormone from days 10-90, and from days 70-90 of preg- 
nancy also were determined for each gilt, and each of these correlated 
with measures of mammary development (Appendix I and II). Hormone concen- 
trations from days 10 through 90 were, in general, less highly correlated 
with mammary development than the mean concentrations from days 10-110 
(compare table IV-7 and Appendix I) . Two exceptions were the correlation 
between E-SO, and mammary wet weight, and the correlation between E-SO, 
and RNA to DNA ratio, which had a higher probability of significance for 
the day 10-90 mean (Appendix I) than for the day 10-110 mean (table IV-7). 

Correlations between mean hormonal concentrations from days 70-90 
and various mammary variables (Appendix II) were less for almost all 



-129- 



associations compared to table IV- 7 . However, correlations were still 
significant and positive for all E-SO, correlations, and highly signifi- 
cant for E,, and DNA concentration (Appendix II)- It was surprising that 
the correlation between the mean of three E„ measurements and DNA con- 
centration was so significant given the variation between E„ samples 
(see mean square error in table IV-3) . Nevertheless, it was apparent 
that mean hormonal concentrations from days 70-90 or from days 10-90 were 
no more highly associated with mammary development than mean concentration 
between days 10 and 110 (table IV- 7 , Appendix I, Appendix II). 

Because pseudopregnant gilts with greatly reduced mammary develop- 
ment also had much lower E-SO, , E , and E„ concentrations, the among 
animal correlations between mean hormonal concentrations and mammary 
development may have been artificially high. Although conceptuses pro- 
duce estrogens and induce mammary development, estrogens and mammary 
development may not be necessarily causally related. To examine this 
in a more discriminating fashion, within-group correlations between mean 
hormone concentrations (days 10-110) and mammary variables were deter- 
mined (Appendix IV) . Within-group correlations for the nine statis- 
tically significant associations in table IV-7 then were tested to 
determine if the coefficients originated from a common rho, and if so, 
were pooled (Snedecor and Cochran, 1969). All pooled within-group cor- 
relations were positive (table IV-8) . Due to loss in degrees of freedom, 

coefficients had to be .667 to be significant at the 5% level, but E-SO, 

4 

still was associated significantly with DNA and RNA concentrations, and 
E„ still was correlated significantly with DNA concentration (table 
IV-8) . Correlation coefficients for E and DNA concentration or for 
prolactin and mammary gland wet weight were high but not significant 



-130- 



Table IV-8. POOLED WITHIN-GROUP CORRELATIONS BETWEEN MEAN HORMONE CON- 
CENTRATIONS AND MAMMARY VARIABLES IN GILTS WITH 0, 4-7, OR 
8-11 FETUSES 



Estrogen- 
Response^ Sulfate Estrone Estradiol Prolactin 



Mammary wet weight .121 .495 

DNA (ug/g DFFT) .709* .517 .740* 

Total DNA .393 

RNA (yg/g DFFT) .677* .159 

Total RNA .312 

*P < .05 

^df = 7. 



-131- 



(P < .05). Interestingly, correlations between prolactin and mammary 
gland wet weight were high for groups 2 and 3 (Appendix III) , but essen- 
tially zero for group 1. Either conceptus-produced estrogen or other 
products were necessary to induce the prolactin receptor, or insufficient 
epithelial tissue was present in group 1 gilts for prolactin to exert the 
effect. 

One must conclude from this experiment that products of the con- 
ceptus do influence mammary development. Among-animal correlation for 
E-SO, and total placental weight was .943 (P < .001), while that for 
E-SO, and total mammary DNA was .721 (P < .01). Free estrone was cor- 
related with the number of fetuses (r = .719, P < .01), and with mammary 
DNA concentration (r = .581, P < .05). While free E„ was not correlated 
with fetal or placental development, primarily due to variability be- 
tween samples, it was correlated with mammary DNA concentration (r = 
.730, P < .01). Nagasawa and Yanai (1971) correlated total placental 
weight with mammary DNA (r = .46, P < .001) and mammary RNA (r = .79, 
P < .001) in mice and concluded that the placental mammogen in mice was 
more lactogenic than mammogenic. A comparison of the among animal or 
pooled within-group correlations in the current experiment suggested 
that estrogens were more mammogenic as measured by DNA and DFFT than 
lactogenic as measured by RNA or RNA: DNA ratio. This probably reflected 
a difference in type of hormone, that is, murine placental lactogen 
versus an estrogen in the pig. 

It was interesting that correlations between E-SO. and mammary 
development were greater, in general, than correlations for free steroid 
hormones. Free E and E^ are biologically active and could be used 
directly by mammary glands. Conversely the conjugated estrogens 



-132- 



presumably would have to be cleaved before being able to bind to a re- 
ceptor. This suggested the presence of sulfatase activity (Ainsworth, 
1972) within the mammary gland. On the other hand, the E-SO^ assay 
utilized in the current experiment measured estrogen-glucuronides as 
well as estrogen sulfate. Therefore, the conjugated estrogens may have 
reflected a large and relatively stable pool of estrogen in which the 
size was dependent upon turnover rate of free estrogen in the extra- 
cellular fluid. Examination of sulfatase activity within the mammary 
gland, quantification of free estrogen turnover rates during gestation, 
and characterization of the conjugated estrogen pool would shed light 
on this area. 



B. Effect of Prostaglandin ^2'^ on Lactogenesis in 
Pseudopregnant Gilts 



Introduction 

In mammals, decreasing concentrations of progesterone are generally 
recognized as the "lactogenic trigger" (Kuhn, 1977) along with increasing 
concentrations of estrogen, prolactin, and glucocorticoids (Tucker, 
1974). Previous research (Knight et al., 1977; Chapter IV, Section A) 
demonstrated that a major portion of estrogen in the peripheral plasma 
of the pregnant gilt during late gestation is of conceptus origin. While 
there is no indication in the present study (Appendix I) that estrogen 
affects prolactin secretion in the pig, it does induce prolactin secre- 
tion in rats (Neil, 1974) and sheep (Vician et al., 1979). It also has 
been shown that fetal Cortisol secretion is the signal to initiate the 
process of parturition in sheep (Liggins, 1972). While not conclusive. 



-133- 



there is evidence to suggest that fetal Cortisol also regulates parturi- 
tion in the pig (Nara and First, 1981a). Therefore, if the pig is similar 
to these other species, porcine conceptuses have the ability to secrete 
or to initiate secretion of hormones of the lactogenic complex. 

Recent experiments suggest that prostaglandin Fa (PGF ct) plays an 
integral role in spontaneous parturition in the pig (Nara and First, 
1981b). In 1974, Diehl et al. showed that PGF a injections induced 
parturition in pregnant gilts by regressing the corpora lutea. They 
further reported that PGF„a induced the presence of secretion within the 
mammary glands (Diehl et al., 1974). Whether the ability of PGF„a to 
induce mammary secretion was simply due to decreased sertim concentrations 
of progesterone, stimulation of prolactin secretion (Renegar et al. , 1978) 
or glucocorticoid secretion (Collier et al. , 1979), or due to a direct 
effect on the mammary gland (Spooner et al., 1977; Rillema, 1980) is 
not known. Lactogenesis can be induced in pregnant rats (Bussman and 
Deis, 1979) and pregnant rabbits (Deis et al., 1980) by injection of 
PGF a. However, it was impossible to deduce from these studies if there 
was a f etal-placental contribution to the differentiation of the mammary 
glands, as defined by increased lactose synthetase, casein synthesis, 
and secretory response. 

In the previous study (Chapter IV, Section A) it was determined 
that mammary gland DNA concentrations in pseudopregnant gilts were only 
22.2% of those from pregnant animals (table IV-3) . While histological 
examination of mammary tissue from these gilts also showed reduced 
epithelial cell numbers (figure IV-6) , the lobulo-alveolar structure 
in mammary glands from pseudopregnant gilts appeared normal. Taking 
the reduced cell numbers into consideration, it was believed that the 



-134- 



pseudopregnant gilt could be used as a model to test, in a preliminary 
fashion, the effect of PGF a on differentiation of the porcine mammary 
gland in the absence of f etal-placental units. 



Materials and Methods 

Five crossbred gilts of similar weight, age, and genetic background 
were injected with 5 mg estradiol-valerate per day on days 11-15 of the 
estrous cycle to induce pseudopregnancy. All animals were kept under 
identical conditions in confinement. On day 108 of pseudopregnancy 
three of the gilts received intramuscular injections of 10 mg (free acid 
equivalent) PGF ct (THAM salt, lutalyse; Upjohn Co., Kalamazoo, MI) at 
0800 and 2000 hr. The remaining two gilts received injections of saline 
at 0800 and 2000 hr. Forty-eight hours after the initial injection of 
PGF a or saline, a single blood sample was collected and placed in a 
heparinized tube; and a biopsy of approximately 5 g was obtained from 
the second most posterior mammary gland of each animal. In addition, a 
laparotomy was performed on each gilt to visually assess the condition 
of the corpora lutea. Blood samples were centrifuged at 8,000 g for 
15 min and the resulting plasma stored at -10 C until analyzed for pro- 
gesterone radio innnunoassay was performed (see Chapter IV, Section A). 

Approximately 2 g of mammary tissue was fixed in Bouin's fixative 
for histological examination, and the remaining 3 g was placed in an 
i ice-cold isotonic tris-sucrose solution (pH 7.3) for studies of metabolic 

I activity. After transporting the tissue to the lab, 100-150 mg slices 

of mammary tissue were prepared with a Stadie-Riggs hand microtome and 
incubated in Krebs-Ringer bicarbonate-buffered solutions containing 



-135- 



either radioactive acetate or glucose (see Chapter III, Section A) to 
determine rates of substrate oxidation or incorporation into lipid. 
Three incubations per substrate per gilt and three blank incubations 
containing no tissue were performed concurrently to determine background 
rates of oxidation or lipid synthesis. Mammary tissue for histological 
examination was dehydrated, embedded in paraffin, and 6-7 y sections 
prepared. After mounting, sections were stained with hematoxylin and 
eosin (Chapter III, Section A) and examined with a Leitz microscope. 
A simple one-way analysis of variance was performed to examine 
the effect of treatment on plasma progesterone concentrations. In the 
tissue slice incubation study, multiple observations were made for each 
animal, and gilts were nested within treatments. Thus an analysis of 
variance was performed where effects of treatment and gilt within treat- 
ment were examined, and the treatment effect was tested against gilt. 

Results and Discussion 

At the time of mammary biopsy, visual appraisal of ovaries indi- 
cated that regression of corpora lutea had occurred in the PGF2a- 
injected animals but not control gilts. Results of the progesterone 
assay indicated that saline injected gilts averaged 10.9 ng progesterone 
per ml as compared to 1.9 ng per ml in PGF a-injected animals (P > .05). 
Failure to detect treatment differences was probably due to low numbers 
of animals used. Collectively, low progesterone concentrations and 
visual indication of corpora lutea indicated that luteolysis was well 
underway in PGF„a-injected animals. This agreed with previous reports 
(Diehl et al., 1974; Nara and First, 1981b) that PGF a was luteolytic in 
the pig at the end of pregnancy. 



-136- 



Histological comparison of mammary tissue from control and PGF a- 
treated gilts was made in figure IV-7 . Several lobules of mammary 
alveoli, clusters of white adipocytes, and a few broad bands of con- 
nective tissue were evident in tissue from both PGF a-injected and 
control gilts (figure lV-7) . More adipose and connective tissue were 
present in the photomicrograph of mammary tissue from a control gilt 
(figure IV-7, A). This was due partly to the increased size of the 
alveolar lumina in PGF a-injected gilts (figure IV-7,B). Secretory 
material was evident in alveolar lumina of both control and PGF^a- 
injected gilts. Alveoli in the mammary glands of saline- injected gilts 
were rounder and more homogeneous in appearance than the more distended 
alveoli of PGF a-injected gilts (figure IV-7). At higher magnification, 
differences between tissues from PGF„ct versus control gilts were even 
more evident (figure IV-8) . Densely staining nuclei were evident in 
the cuboidal-shaped epithelial cells of alveoli from control gilts 
(figure IV-8, A). The alveoli had a rather rounded appearance and the 
secretions stained uniformly within the lumina. In contrast, at the 
same magnification, mammary tissue from PGF„a-treated gilts showed 
alveoli which appeared to be more differentiated (figure IV-8,B). Since 
milk removal had not occurred. Increased secretion within the alveolar 
lumina had forced the epithelial cells back against the basement mem- 
brane so that they took on a more squamous appearance. Secretions 
within the lumina did not stain homogeneously but showed numerous lipid 
droplets. Furthermore, large accumulations of lipid were evident near 
the apical ends of certain epithelial cells (figure IV-8,B) suggesting, 
that at only 48 hr after PGF a injection, cells were active in milk-fat 
synthesis and secretion. 



Figure IV-7. Histological photomicrographs of mammary tissue from 

control (A) or prostaglandin F2a-injected gilts (B) on 
day 110 of pseudopregnancy. Magnification X16. 



-138- 



*7^ 



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f*v-*~'<ai'J'>^ - - ~ 



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1l ,, 










Figure IV-8. Histological photomicrographs of mammary tissue from 

control (A) or prostaglandin F2a-injected gilts (B) on 
day 110 of pseudopregnancy. Magnification X160. 



-140- 



' n.» 



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-141- 



A summary of the tissue slice incubation studies is in tables IV-9 
and IV-10. Mammary tissue from control animals oxidized 374 nmoles 
acetate per 100 mg tissue per 3 hr, whereas tissue from PGF a-treated 
gilts oxidized 565 nmoles per 100 mg per 3 hr incubation. Rates of 
glucose oxidation by 100 mg mammary tissue were 110 and 137 nmoles for 
control and treated gilts, respectively. Analysis of variance of these 
results (table IV-10) indicated that neither gilt nor treatment had a 
significant effect on acetate oxidation, but that gilt affected rates 
of glucose oxidation (P < .01). Rates of two-carbon unit (TCU) oxida- 
tion (figure IV-9) averaged 704 nmoles per 100 mg per 3 hr for control 
gilts and 991 nmoles per 100 mg for PGF a-treated animals. While there 
was a trend for increased oxidation by tissue from PGF a-treated animals, 
differences were not significant (table IV-10) undoubtably due to the 
low numbers of animals. 

Rates of acetate incorporation into fatty acids averaged 162 nmoles 
per 100 mg per 3 hr incubation in saline- injected gilts, and 394 nmoles 
per 100 mg in PGF a-treated animals (table IV-9), and both gilt (P < .05) 
and treatment (P < . 10) were significant (table IV-10) . Rates of glu- 
cose incorporation into fatty acids were very low for both treatment 
groups (table IV-9) , and the groups did not differ (table IV-10) . The 
combined measure of acetate and glucose incorporation into lipid, TCU 
incorporation, is shown in figure IV-10. Results suggested a niimerical 
but not a statistically different trend between the two treatment groups 
(table IV-10) . 

There was a consistent trend for metabolic activity to be higher 
in tissue from PGF^a-treated gilts than in tissue from saline-injected 
control gilts. This was expected since the histological responses 



■142- 



Table IV-9. MEAN RATES OF SUBSTRATE OXIDATION AND INCORPORATION INTO 
LIPID BY MAMMARY TISSUE SLICES IN VITRO 



Treatment Group 



Control Prostaglandin Fa 



Acetate oxidation 374 ± 92 (3) 565 ± 53 (9) 

Glucose oxidation 110 ± 37 (3) 137 ± 22 (9) 

Acetate incorporation 162 ± 72 (6) 394 ± 58 (9) 

Glucose incorporation 22 ± 7 (6) 9 ± 6 (9) 



All values represent mean ± SEM in nmoles substrate per 100 mg tissue 
per 3 hr incubation. The number of observations is indicated in 
parentheses. 



-143- 







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-148- 



indicated that lactogenesis had ensued In the PGF a-treated gilt. How- 
ever, the low numbers of animals, and the limited numbers of secretory 
cells present within the pseudopregnant mammary gland, made it difficult 
to detect significant treatment effects. Rates of activity observed in 
mammary tissue from PGF„a-injected animals were similar to rates pre- 
viously observed in gilts between days 90 and 105 of pregnancy (Chapter 
III, Section A), while rates of activity of control animals were similar 
to those observed earlier in pregnancy (days 30, 45). 

Despite the limited numbers of gilts utilized in this study, it is 
probably safe to say that the pig is not unlike the rabbit (Deis et al., 
1980) or rat (Bussman and Deis, 1979) in which PGF a injections elicit 
a response characteristic of lactogenesis. 

Since no literature was available indicating metabolic activity of 
pseudopregnant porcine mammary tissue, there was nothing to compare 
those in the present study with. However, the histological appearance 
of tissue from PGF a-injected versus control gilts leaves no doubt of 
a lactogenic response to the prostaglandin. Whether PGF.a exerts a 
direct effect on mammary tissue or an indirect one thru alteration of 
other hormones such as progesterone, prolactin, or glucocorticoids re- 
mains to be established. 

C. Summary 

Experiments were conducted to examine effect of conceptuses on 
mammary gland growth and lactogenesis in the pig. In the first experi- 
ment concentrations and profiles of conjugated estrogens, E , E„, pro- 
gesterone, and prolactin, as well as measures of mammary gland development. 



-149- 



were characterized in gilts with to 11 conceptuses. In general, pro- 
files of hormones in pregnant gilts were similar to those previously 
reported. While concentrations of E. , progesterone, and prolactin were 
similar to previous reports, concentrations of the conjugated estrogens 
and E„ were higher than previously observed, possibly due to assay dif- 
ferences. Concentrations of E-SO,, E, , and probably E^. but not pro- 
gesterone or prolactin, were associated with or to fetal numbers, total 
fetal weight, total placental weight, and empty uterine weight (table 
IV-4) . In contrast, only progesterone was highly correlated with number 
of corpora lutea. Results suggested that the conjugated estrogens, E^, 
and much of E were probably of fetal-placental origin, whereas little, 
if any, placental production of prolactin or progesterone occurs. Maimnary 
gland measurements Indicated that glands from pseudopregnant gilts had 
only 22-23% of the DNA and RNA observed in glands from pregnant animals, 
but amounts of mammary nucleic acids were not proportional to conceptus 
numbers in pregnant gilts. 

Degree of mammary development in pseudopregnant rats (Desjardins 
et al., 1968) was similar to that observed in pseudopregnant gilts. In 
the rat maternal hormones were primarily responsible for mammary gland 
development in the first half of pregnancy, but hormones from the fetal- 
placental units become the primary limiting factors for mammary gland 
growth during the second half of gestation (Desjardins et al., 1968). 
Since conceptus presence was required for normal mammary development at 
day 110 of pregnancy this hypothesis may also be true in the pig. How- 
ever, the pig clearly is unlike the mouse in which mammary gland DNA 
was linearly related to conceptus number (Nagasawa and Yanai, 1971). 
The mouse and rat produce a placental lactogen, whereas the pig does 



-150- 



not (Talamantes et al. , 1980). However, concentrations of E-SO , E^ , and 
E were clearly related to conceptus development (table IV-3) and mammary 
development (tables IV-7, IV-8) in the pig, and concentrations of these 
estrogens increased between days 70 and 90 of pregnancy (figures IV-1, 
IV-2 , IV-3). This corresponds to the time when a large increase in mammary 
DNA was observed (figure III-3) , suggesting that estrogen was the pla- 
cental mammogen in the pig. Mechanism of action of estrogen on mammo- 
genesis probably was not via stimulation of prolactin secretion since 
no increase in concentration of prolactin was observed in pregnant compared 
to pseudopregnant gilts. However, it would be interesting to know if 
estrogen induced and regulated the prolactin receptor in the porcine mam- 
mary gland. If the action of estrogen on mammogenesis was indirect, as 
in the induction of the prolactin receptor, then one would not expect to 
observe a linear relationship between conceptus numbers and mammary gland 
DNA (as seen in the mouse) , but a response more like that observed in 
the pig. Evidence to support this hypothesis was provided by within- 
group correlations (Appendix III) . Large positive correlations existed 
between prolactin concentration and mammary wet weight in group 2 and 3 
gilts which had high concentrations of estrogen. In contrast, the cor- 
relation between prolactin and mammary wet weight was essentially zero 
in group 1 animals with low concentrations of estrogen (Appendix III) . 

A second study was performed to determine whether lactogenesis could 
be induced in pseudopregnant gilts in absence of conceptuses, and there- 
fore absence of the hormones produced by chronically fetal-placental 
units. Prostaglandin Fa (PGF„a) was utilized to regress corpora lutea 
since there was evidence to suggest that it plays a role in spontaneous 
parturition (Nara and First, 1981b). Forty-eight hours after injection 



-151- 



of PGF a, luteal regression and a decrease in plasma progesterone were 
observed in PGF a-injected, but not control, gilts. Mammary tissue from 
PGF a-injected animals was more differentiated than tissue from control 
animals, with increased lipid and secretion within the alveolar lumina, 
and a squamous appearance of epithelial cells surrounding the distended 
alveoli (figures IV-7, IV-8) . Rates of oxidation and fatty acid syn- 



I 



thesis by mammary tissue slices in short term incubation were marginally, [ 

but consistently, greater in PGF a-injected animals than in control [ 

gilts (table IV-9) . However, due to the low numbers of animals avail- ; 

t 

able, and due to the reduced number of epithelial cells in the pseudo- 
pregnant mammary gland, these differences were not significant, Col- 

l 

lectively, the histology and tissue slice incubation results, although [ 

i 

preliminary in nature, suggested that some epithelial cell differentiation > 

I 

and therefore lactogenesis could be induced in the pseudopregnant gilt ; 

i 
by injections of PGF a. However, the reduced DNA in the pseudopregnant I 

mammary glands did not allow a direct comparison with tissue from gilts 

during lactogenesis following a normal pregnancy. The positive effect 

observed by injection of PGF a suggests one might conduct further studies 

in which PGF a would be used in pregnant gilts (at various times pre- 

partum) to determine the precise influence of the prepartum estrogen 

rise on lactogenesis. 



•r^t^faiT'Sii: ■ 



CHAPTER V 
PRODUCTION AND ISOLATION OF BOVINE PLACENTAL LACTOGEN 

A. Introduction 

Several lines of evidence indicated a positive relationship between 
conceptus and mammary development in dairy cattle. Records, from over 
1700 parturitions at the Florida Agricultural Experiment Station, in- 
dicated that calf birth weight was related to subsequent milk yield of 
the dam (Erb et al. , 1980; Thatcher et al., 1980). Fetal weight and 
maternal mammary gland development during pregnancy (as measured by duct 
surface area) are essentially parallel (Thatcher et al., 1980). This 
may suggest that the fetus regulates growth and therefore the functional 
capacity of the mammary gland, or that development of both are regulated 
by common substances. Evidence that the bovine placenta produced a 
lactogenic substance was provided by co-culture of cotyledons and maimnary 
tissue explants (Buttle and Forsyth, 1976) , and by cross-reactivity 
of bovine placental extracts with human placental lactogen antiserum 
(Gusdon et al. , 1970) . 

The first report of purification of bovine placental lactogen 
(bPL) was in 1976 (Bolander and Fellows). The latter reported that 
placental lactogen concentrations during pregnancy were related to sub- 
sequent milk yield in dairy and beef cows (Bolander et al., 1976). This 
was of great significance to the dairy industry because it showed promise 



-152- 



-153- 



as a selection tool for breeding animals, and because it had identified 
the heretofore hypothesized placental mammogen. Placental lactogen 
concentrations were also positively related to both litter size and milk 
yield in goats (Hayden et al. , 1979) and sheep (Butler et al. , 1981). 
Curiously, several subsequent reports on isolation of bPL (table V-1) 
have indicated quite different molecular weights and biological poten- 
cies than originally reported by Bolander and Fellows (1976) , and there 
was little agreement between any of the reports. This created con- 
siderable uncertainty as to the existence, structure, and biological 
function of bovine placental lactogen. Although, Bolander et al. (1976) 
reported concentrations of greater than 1000 ng per ml of plasma using 
a radioimmunoassay developed against their preparation, Kelly et al. 
(1973), Roy et al. (1977), Hayden and Forsyth (1979), and Schellenberg 
and Friesen (1981) indicated that concentrations of bovine placental 
lactogen in maternal blood remained below 150 ng/ml at all stages of 
gestation. Clearly, there were numerous physical differences between 
the various preparations of bPL (table V-1) , and a definite need for 
clarification of molecular weight and biological activity of authentic 
bPL before questions about the physiological role(s) of the placental 
polypeptide could be addressed. 

The general approach taken to isolate bPL in the aforementioned 
studies (acknowledging that methods are brief since three of the reports 
in table V-1 are abstracts) was to homogenize cotyledons in buffer or 
organic solvent followed by extraction with ammonium bicarbonate 
(Bolander and Fellows, 1976; Beckers et al. , 1980). Resulting extracts 
had .67 (Beckers et al., 1980) and .032 (Bolander and Fellows, 1976) pg 
prolactin equivalents per mg protein. Because of relative impurity of 



-154- 



Table V-1. PREVIOUS REPORTS OF THE ISOLATION OF BOVINE PLACENTAL 
LACTOGEN 



Study 



M Wt 



pi Biological Potency 



1 Bolander and Fellows (1976) 22150 

2 Roy et al. (1977) 60000 

3 Hayden and Forsyth (1979) 45000 

4 Bremel et al. (1979) 32000 

5 Beckers et al. (1980) 



5.9 



5.3 



0.04 lU/mg" 
7.1 lU/mg^ 
25.0 lU/mg'^ 



16.1 lU/mg'^ 



Measured by radioreceptor assay. 
Measured by mammary gland bioassay. 



-155- 



initial extracts, and obvious differences between preparations among 
laboratories (table V-1) it seemed that bovine cotyledonary culture 
might be a useful technique. In this procedure only bPL and other 
secretory proteins would be discharged into the medium. By centri- 
fuging down the tissue and recovering the supernatant, structural pro- 
teins and various other contaminating cellular macromolecules would be 
removed at the onset. Indeed, this approach had been utilized pre- 
viously by Swanson and Bremel (1980) to produce bPL in vitro. Thus, 
reducing contamination by undesirable proteins should enhance purifica- 
tion. 

B. Materials and Methods 

Four Jersey cows in the last trimester of pregnancy (day 231, 
236, 253, and 276) were slaughtered to provide cotyledons for culture. 
As soon as possible after the animal was stunned (usually within 20 min) , 
the entire uterus was removed intact and transported to the laboratory 
for dissection. Under sterile conditions several placentomes were 
excised from the uterus, the cotyledons were gently pulled away from the 
caruncles and cotyledonary tissue placed in ice-cold, minimal essential 
medium. Cotyledonary explants of approximately 5-10 mg were prepared 
from the cotyledons under a laminar flow hood taking care to minimize 
amount of connective tissue present. Approximately 700 mg of explants 
were placed into each 100 x 15 mm petrie dish (Falcon Plastics, Oxnard, 
CA) along with 15 ml of modified minimal essential medium (see Appendix 
V) supplemented with nonessential amino acids, glucose (5 g/1) , insulin 
(200 mU/ml) , L (4,5- H) leucine (60 Ci/mmole; Schwarz Mann, Orangeburg, 



-156- 



NY) , penicillin (100 U/ml) , streptomycin (100 U/ml) , and antimycotic 

3 

(.25 yg/ml) . Five to ten yCi H-leucine generally was added to each 

3 
culture dish, but two culture dishes received 100 uCi H-leucine and 

only 10% of the normal leucine concentration to enhance the incorpora- 
tion of the label into secreted proteins. All tissue culture supplies 
were purchased from Grand Island Biological Co. (Grand Island, NY). 
When all culture dishes were prepared they were placed into a controlled 
atmosphere chamber (Bellco Glass Co., Vineland, NJ) which was placed 
on a rocker platform (Bellco Glass Co.) set at 6 cycles per minute, in 
a 37° chamber. The atmosphere chamber was gassed every 12 hr with 
N :0 :C0 (50:45:5). After 24-36 hr of incubation tissue was separated 
from the media by centrifugation at 6000 g for 10 min. The supernatant, 
containing the secretory protein, was placed into dialysis bags (molecu- 
lar weight cut off less than 3500) and dialyzed for 36 hr against 10 mM 

Tris-HCl buffer, pH 8.2, to remove the salts and other low molecular 

3 
weight components of the media (including unincorporated H-leucine). 

Since at least four cultures were performed at one time, and since the 
stability of bPL was a major concern, the dialyzed medium then was fast- 
frozen and lyophilized for storage at -10 C. 

In order to monitor purification of bPL it was necessary to have 
a "biological assay" to distinguish it from other proteins. The assay 
selected was the rabbit mammary gland lactogenic radioreceptor assay 
which has been used extensively for purification of placental lactogens 
in other species (Roy and Friesen, 1979). The procedure for preparation 
of the microsomal membranes used as the receptor membrane preparation 
was identical to that of Shiu et al. (1973) with the exception that 
ergocryptine (4 mg) was injected into does on days 8 and 9 of lactation 



-157- 



] to Increase the number of unoccupied receptors (Durand and Djiane, 1977). 

.J 

j Details of the procedure utilized (buffers, injections, and method) are 

■I 

I given in Appendix VI. 

lodination of prolactin (NIAMDD-oPRL-14) for radioreceptor assay 

was accomplished using lodo-gen reagent (Pierce Chemical Co., Rockford, 

XL) in 25 mM gris buffer, pH 7.20. Five micrograms of hormone (5 ug/10 ul) 

125 
and 1 mCi of Na I (1 mCi/10 yl) were added to a 12 x 75 borosilicate 

tube which was coated with 2 yg lodo-gen (70 yl reaction volume) and the 

reaction allowed to proceed for 15 min. lodinated hormone and free 

iodine were subsequently separated on a colimin (approximately .7 x 15 cm) 

of Biogel P-60 (RioRad Laboratories) . While protein determinations were 

not performed on the lodinated hormone, minimum specific activity 

achieved (that is, the specific activity if all 5 yg of hormone were in 

the peak tube selected for radioreceptor assay, and based on an average 

of 213 X 10 cpm in the peak tube) was 19.4 Ci per ug hormone which cor- 

125 
responds to 1 ng of I-prolactin (50,000 cpm) added to each tube. 

Since the assay was not sensitive below 10 ng, this was adequate spe- 
cific activity. 

The procedure utilized for the radioreceptor assay was based on 
that of Shiu et al. (1973) with exception that the incubation occurred 
at 5 C for 24-48 hr (which maximized specific binding) . The procedure 
and validation are summarized in Appendix VII and was performed by 
Charles R. Wallace (unpublished) . Briefly, the validation indicated 
that bovine LH, FSH, insulin, and thyroxine did not cross react at 
1000 ng/assay tube in the system. Cross-reactivities of bovine growth 
hormone and human placental lactogen were 4.3 and 3.2%, respectively. 
Specificity of the assay was very similar to that of Shiu et al. (1973) 



-158- 



except for the low cross-reactivity of human placental lactogen, which 
was equipotent to prolactin in their hands. In 27 radioreceptor assays 
performed during the course of bPL purification average binding in the 
absence of cold hormone was 15.1%, and 72.7% of that was displaced by 
500 ng of prolactin (NIH-PB4) standard. 

Two-dimensional polyacrylamide gel electrophoresis of culture media 
from cotyledonary cultures was performed by Freida Sessions as described 
by Horst and Roberts (1979). One milligram of crude or partially puri- 
fied lyophilized protein was dissolved in 1 ml 5 mM K CO containing 
9.4 M urea. 2% (V/V) Nonidet P-40, and 0.5% (W/V) dithiothreitol. One- 
hundred microliters of this solution (containing 100 ug protein) was 
subjected to isoelectric focusing in the first dimension in 4% (W/V) 
acrylamide gels which contained N,N'diallyltartardlamide, urea (9.4 M) , 
Nonidet P-40 (2%, V/V) , and 2% (V/V) ampholines (50% pH 3.5-10; 35% 
pH 5-7; and 15% pH 9-11). After isoelectric focusing the gels were 
equilibrated in 65 mM Tris-HCl, pH 6.9, which contained 1% (W/V) sodium 
dodecyl sulfate and 1% (V/V) 2-mercaptoethanol. Then the gels were 
overlaid on 10% (W/V) acrylamide slab gels and electrophoresis performed 
toward the anode. After completion of electrophoresis slabs were fixed 
in acetic acid:ethanol (7:40), and stained with Coomassie Blue R-250. 
Coomassie Blue R-250 and N,N'diallyltartardiamide were purchased from 
Bio-Rad laboratires (Richmond, CA) . Sodium dodecyl sulfate was obtained 
from BDH Chemicals Ltd. (Poole, England). Ampholines came from LKB 
(Uppsala, Sweden) and amino acids, dithiothreitol, and 2-mercaptoethanol 
were from Sigma Chemical Co. (St. Louis, MO). Urea was from Pierce 
Chemicals (Rockford, XL). 



-159- 



The standard procedure to purify bPL in the current study was 
initiated by dissolving 41-200 mg of dialyzed, lyophilized cotyledon 
culture material in 2-4 ml of 25 mM Tris-HCl, pH 8.2 containing .2 M 
NaCl. In addition, .02% sodium-azide was added to all buffers used 
during purification. The .2 M NaCl was necessary to prevent aggrega- 
tion of proteins, which also had been observed by Bolander and Fellows 

(1976) for bPL and by Hurley et al. (1976) for ovine placental lactogen. 
After crude material was dissolved, it was applied to a Sephacryl S-200 

(Pharmacia Fine Chemicals, Piscataway, NJ) column (1.5 x 90 cm) which 
was pre-equilibrated with the same buffer. Subsequently, 80 fractions 

(2 ml) were collected, and a portion of each fraction was utilized for 

3 
protein assay (Lowry et al. , 1951), H analysis, and for lactogenic 

radioreceptor assay. Two of the more concentrated crude preparations 
did not dissolve completely in the 25 mM Tris-HCl buffer and were centri- 
fuged to remove the insoluble material prior to loading on the column. 
f Fractions from the S-200 column which were most active in the radio- 

receptor assay were pooled together and dialyzed for 36 to 48 hr against 
three changes of 10 mM Tris-HCl, pH 8.2 to remove all the NaCl. The 
dialyzed fractions (approximately 20 ml) then were applied to a 
diethylaminoethyl-cellulose (Whatman, Inc., Clifton, NJ) column (1.5 x 
10 cm) equilibrated in 10 mM Tris-HCl, pH 8.2. After washing the column 
with 30-40 ml of the equilibration buffer, a 2 x 150 ml to .5 M NaCl 
gradient was begun. Fractions of 2.4 ml were collected until the gra- 
dient was completed. As before, aliquots of each fraction were removed 

3 
for H, protein, and lactogenic activity determination. The diethylamino- 
ethyl-cellulose (DEAE) fractions most active in the radioreceptor assay 
were pooled and again dialyzed for at least 24 hr against 10 mM Tris-HCl, 



-160- 



pH 8.2. Finally, lyophilization was performed to concentrate the material 
prior to gel filtration with Sephadex G-75 (Pharmacia Fine Chemicals). 

The G-75 column (1.5 x 58 cm) was equilibrated in, and the lyo- 
philized DEAE-cellulose active fractions were dissolved in the same 

buffer used for S-200 chromatography. After loading the sample, 2.0 ml 

• 3 

fractions (n = 50) were collected and monitored for H and lactogenic 

activity. Both the S-200 and G-75 columns were standardized with at 
least three proteins of known molecular weight prior to use. Details 
for construction of the selectivity curves are in Appendix VIII. 

During the course of the present study, purification was attempted 
on four batches of cotyledon culture proteins. The first batch was pro- 
cessed through the S-200 and DEAE-cellulose columns. Since radioactivity 
was quite low, all of the active DEAE material was pooled, lyophilized 
and used for two-dimensional polyacrylamide gel electrophoresis (2-D 

PAGE) , and subsequent f luorography. The second batch of material (from 

3 

the 100 yCi H-leucine cultures) was processed up to the G-75 column. 

However, all of the lactogenic activity was lost when the fraction col- 
lector malfunctioned. The third batch of protein (from cultures sup- 
plemented with 10 yCi H-leucine) was purified through the S-200, DEAE- 
cellulose and G-75 columns, and the partially purified bPL was tested in 
a mammary tissue explant culture bioassay. To accomplish this, a por- 
tion of the pooled active fractions from the G-75 column (equivalent to 
180 yg prolactin) was lialyzed for 48 hr against several changes of 1 mM 
Tris-HCl to remove salt and (especially) the sodiiom-azide preservative. 
The bPL preparation then was lyophilized, and based on radioreceptor 
assay of the pooled G-75 fractions was redissolved in 1.80 ml of physio- 
logical saline to achieve a concentration of 100 yg prolactin-equivalents 



-161- 



(based on NIH-PB4) per ml. This concentration was necessary for addi- 
tion of bPL to culture media without extensively diluting the media. 

The fourth batch of bPL contained the remainder of secretory protein 

3 
from cultures supplemented with 100 yCi H- leucine. This latter protein 

was purified through the G-75 step in the procedure, and fractionated 

further through a column of Cibicron-Blue (obtained from Michael Horst 

of the Microbiology and Cell Science Department), since this material , 

had been used effectively by Beckers et al. (1979) in their studies on 

bPL. The protein was dialyzed against, and the column equilibrated with - 

20 mM Tris-HCl, pH 8.1, containing .005 M MgCl as in Beckers et al. i 

i 
(1979). After application of the sample, a to .3 M KCl gradient |: 

(2 X 75 ml) was performed while collecting 90 fractions of 2.4 ml each. 

To test the biological activity of partially purified bPL after 
fractionation by Sephacryl S-200, DEAE-cellulose, and Sephadex G-75 
chromatography, a rabbit mammary gland explant culture bioassay was 
performed. Histology and rate of 2- C-acetate (2.0 mCi/mmole; New 
England Nuclear, Boston, MA) incorporation into fatty acids were ex- 
amined in explants prior to culture, in explants cultured for 48 hr in 
medium containing insulin and Cortisol (IC) , in explants cultured for 
48 hr in media containing insulin, Cortisol, and various amounts of pro- 
lactin (ICPrl) , and in explants cultured for 48 hr in medium containing 
insulin, Cortisol, and various amounts of bPL (ICbPL) . The medium used 
was Tissue Culture Medium 199 (Difco Laboratories, Detroit, MI) which was 
supplemented with acetate and glucose to 10 mM, each. In addition, all 
incubation media contained antibiotic-antimycotic (ABAM; Grand Island 
Biological Co., Grand Island, NY), bovine sertrai albumen (BSA, 1 lig/ml) , 
non-essential amino acids (Grand Island Biological Co.), Cortisol 



-162- 



(4-pregnen-llg ,17a,21-trlol-3,20-dione; Steraloids, Inc., Pawling, NY), 
and insulin (Sigma Chemical Co., St. Louis, MO). The insulin (dissolved 
in .005 N HCl) and Cortisol (dissolved in ethanol) were added at 1 ]ig/ml. 
Concentration of ethanol in the final incubation medium was only 1 pl/ml. 
Additions of prolactin (NIAMDD-oPRL-14) in .001 N NaOH to the IC medium 
formed media with 1, 10, 100, 250, 500, or 1000 ng prl/ml. Partially 
purified bPL was added to IC media to provide incubation media with 1, 
10, 100, 250, 500, 1000, and 5000 ng prl-equivalents per ml. Two to 
5 mg explants of mammary tissue from a rabbit pregnant 19 days were 
prepared under an ultraviolet hood and incubated on stainless steel 
grids in plastic culture dishes (Falcon Plastics Co. , Oxnard, CA) con- 
taining 1 ml of incubation medium. A total of 168 culture dishes con- 
taining 2-3 explants each were incubated at 37 C in an atmosphere of 
N -0 rCO^ (50:45:5) for 48 hr (12 dishes each for the 14 different 
media). At the end of the incubation, tissue from three of the 12 
culture dishes from each media treatment group were placed in Bouin's 
solution for fixation. At a later time, tissue was dehydrated and em- 
bedded in paraffin, stained with hematoxylin and eosin, and 7 y sections 
were examined by light microscopy as in Chapter III. The remaining nine 
culture dishes for each medium were utilized for biosynthesis studies. 
Tissue from these nine cultures was distributed among three incubation 
flasks so that each contained 12-38 mg mammary tissue. Three milliliters 
of Krebs-Ringer bicarbonate buffer, pH 7.3, containing 10 mM acetate, 
10 mM glucose, 133 mU insulin/ml, and 2- C-acetate (see Chapter III) 
were placed in 25 ml Erlenmeyer flasks along with explants, gassed with 
:C0 (90:10), and incubated for 2.5 hr in a Dubnoff shaking water bath 
at 37 C. After termination of incubations by addition of 100 \il of 



-163- 



1 N sulfuric acid, fatty acids were saponified, extracted, and quantified 
as in Chapter III, except that only one-half the volume of each reagent 
was utilized since the incubations contained only 50% of the tissue. 
Short term incubations also were performed for tissue on the day the 
culture was begun, that is, tissue at time zero. 

Biosynthesis data were analyzed by least squares regression accord- 
ing to the Statistical Analysis System (Barr et al. , 1976) using the 
models of table V-4. Effects of the three incubation media (IC, ICPrl, 
ICbPL) and the different doses of lactogenic hormone were examined along 
with the medium by dose interaction. In addition, the dose of lactogenic 
hormone was also used as a continuous independent variable to examine 
the polynomial effect of dose after correcting for media effects. 

C. Results and Discussion 

3 
Incorporation of H-leucine into nondialyzable macromolecules, a 

crude measure of biosynthetic activity, was monitored in two of the 

cotyledon cultures by measuring radioactivity in the medium before and 

after dialysis. Cotyledons did synthesize proteins during the culture 



3 • 

period as evidenced by the incorporation of 11.6% of the 100 \iC± of H- i 

leucine in a culture with only one-tenth the normal leucine concentration. 

Cultures performed at the same time, but with the normal concentration 

3 
of leucine (52 mg/1) and 5 \iCi. H-leucine per dish incorporated 6.3% of 

the label added. In addition, lactogenic activities in fresh, dialyzed 

samples of medium, as measured by radioreceptor assay, were 5.2 \ig 

prolactin-equivalents/ml (prl equivalents /ml) and 54.0 yg prl equivalents/ 

ml for cotyledons taken from cows at 253 and 236 days of gestation, 



-164- 



respectively . Whether this difference was due to differences in stage 
of gestation of cows, or variation among individual cotyledons was not 
known since these factors were all confounded. Swanson and Bremel (1980) 
reported that lactogenic activity increased in a linear fashion for 20 
hr in a suspension culture with 100 mg tissue per ml medium, with a peak 
concentration of 40 yg prl equivalents /ml (Sqnason and Bremel, 1980). 
This was in between the concentrations measured in two cultures from 
the current investigation. 

Bremel (University of Wisconsin, Madison) indicated that bPL was 
not stable during freezing (personal communication) . To investigate 
this possibility, a portion of fresh meditmi was assayed, frozen over- 
night, and then thawed and reassayed the following day. Lactogenic 
activity decreased by 21.7% in the sample of medium indicating that 
freezing did induce some conformational change which rendered the poly- 
peptide less active in the radioreceptor assay. Consequently, freezing 
of bPL solutions was avoided during the subsequent purification except 
for fast-freezing (using ethanol and dry CO ) prior to lyophilization. 

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of 
only 100 iJg of cotyledon culture material prior to any purification 
(figure V-1) showed the large number and diverse array of proteins which 
were present, some of which may be of serum origin. While there were 
three or four major proteins present in the slab, one could count as 
many as 40 minor spots in the gel. The most densely staining protein 
in the crude material had a molecular weight of approximately 67,000 and 
a slightly acidic isoelectric point (pi =6.2) which was similar to that 
observed for bovine sertmi albumen in the same system. The other major 
proteins had molecular weights less than 40,000 and also were slightly 







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■167- 



acidic. Almost all proteins visible had molecular weights in the 24,000 
to 140,000 dalton range and the majority were acidic with a pi of less 
than 6.6. This initial characterization of the proteins present hinted 
that fractionation with Sephacryl S-200 followed by DEAE-cellulose would 
be an effective means of separating these proteins. 

A chromatogram of the secreted macromolecules after gel filtration 
with S-200 (figure V-2) indicated that lactogenic activity eluted as a 
broad band with the majority of the peak in the 80,000 and 19,000 dalton 
range. In contrast, there was a broad trailing peak of radioactivity 
which began to elute at the void volume (suggesting a molecular weight 
of approximately 200,000). Consequently, lactogenic activity eluted in 
the trailing shoulder of the radioactivity peak (figure V-2) . The elu- 
tion profile of protein from the S-200 column was more related to radio- 
activity than to the lactogenic activity. While the protein peak was 
not as relatively high as radioactivity in fractions 29-38, they tended 
to be parallel after that, and both indicated a minor protein peak in 
the low molecular weight range (fractions 65-70). The large amount of 
radioactivity prior to the lactogenic peak suggested that the majority 
of the incorporated H-leucine was in proteins much larger than bPL. 
Fractions most active in lactogenic activity were pooled and prepared 
for fractionation on DEAE cellulose. 

Because of the acidic nature of most of the proteins in the unpuri- 
fied cotyledon preparation (figure V-1) , anion exchange chromatography 
with DEAE-cellulose at pH 8.2 was used to fractionate the pooled materal 
from gel-filtration (figure V-3) . Little radioactivity and virtually 
no lactogenic activity eluted during the column wash (figure V-3) . Radio- 
activity began to elute soon after the beginning of the to .5 M NaCl 



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-170- 



gradient and remained elevated through the first 50% of the gradient 

3 
(only to . 35 M gradient shown because all protein and H had eluted 

at that point) . Lactogenic activity eluted soon after radioactivity 

first came off the column, and lactogenic activity had cleared the 

column 33% (mean of four column runs) into the gradient. This indicated 

that most other secretory proteins were more acidic than the lactogenic 

protein (figure V-3) . Most of the protein, as measured by the method 

of Lowry et al. (1951), eluted from the DEAE column soon after bPL and 

paralleled the right hand shoulder of the radioactivity peak. The DEAE 

chromatography verified that bovine cotyledons synthesized ntimerous pro- 

3 

teins in vitro , as indicated by the broad elutlon band of H in figure 

V-3. Beckers et al. (1980) observed bPL to elute from DEAE-Sephadex 
between .075 and .1 M ammonium-bicarbonate which was similar to the 
ionic strength at which bPL eluted in the current study. However, 
Bolander and Fellows (1976) observed bPL to elute from DEAE-cellulose 
in two distinct peaks between .05 and 1.0 M NaCl. 

The DEAE-cellulose active fractions were pooled and gel filtration 
was again performed, but on a coltram of Sephadex G-75 (figure V-4) . The 
lactogenic activity eluted between fractions 19 and 29, with a peak 
corresponding to a molecular weight of approximately 32,000. The peak 
of radioactivity was much broader than this, beginning at the void 
volume (fraction 14) and continuing to fraction 36 (figure V-4) . 

Material from the active Sephadex G-75 fractions was subjected to 
2-D PAGE (figure V-5) . Several groups of proteins at a common range of 
isoelectric points but at molecular weights which ranged from 29,000 
to 100,000 daltons could be detected. That proteins had common iso- 
electric character (pi = 6.2-6.4) was perhaps not surprising since 









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-175- 



position of elution from DEAE cellulose (early in the NaCl gradient, 
see figure V-3) selected for slightly acidic proteins. That families 
of proteins were at such different molecular weights was very surprising 
since final purification procedure prior to 2-D PAGE was Sephadex G-75 
chromatography and fractions pooled from that column eluted in the re- 
gion corresponding to 25,000-40,000 daltons. Examination of figure V-5 
shows seven families of proteins at approximately 29,000, 33,000, 36,000, 
51,000, 61,000, 80,000, and 100,000 daltons, of which the major group 
was the one at 36,000. Only the smallest three were of the size pooled 
from the G-75 column. Closely migrating groups or families of proteins 
(at approximately 22,000 daltons, but different pis) also have been 
observed with human placental lactogen (Chatterjee et al., 1977) and 
with human growth hormone (Lewis et al. , 1980) after isoelectric focusing. 
These groups apparently represent genetic variants (Chatterjee et al. , 
1977). 

In examining the largest proteins, it appeared that either the bPL 
had aggregated (possibly by hydrogen bonding or hydrophobic interactions) , 
or that multiple monomers of bPL had joined by disulfide bonding. 
Dimeric human placental lactogen (Schneider et al. , 1977) is a disul- 
fide-linked dimer of human placental lactogen, which is stable in the 
presence of strong denaturing agents. However, the 2-D PAGE system 
utilized dithlothreitol and mercaptoethanol which were expected to re- 
duce disulfide linkages. In addition, sodium dodecyl sulfate would 
discourage aggregation of proteins. It may be that the smaller two 
families of proteins were fragments or hydrolytic products of proteinase 
action on the native hormone (probably the 36,000 dalton group), and 
that the larger proteins were prohormones of bPL. However, the latter 



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■177- 



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-178- 



suggestion did not seem likely because prohormones are generally only a 
fraction larger than native hormone, and because most of these proteins 
were prestamed to be secreted or of serum origin. 

A summary of the purification of bPL from the secretory material 
of cultured cotyledons (table V-2) demonstrated the advantage of this 
approach as opposed to beginning with cotyledonary homogenates. Initial 
extracts of bPL (derived after homogenization) had .67 (Beckers et al. , 
1980) and .032 (Bolander and Fellows, 1976) yg prl-equivalents/mg pro- 
tein compared to 13.25 Mg/mg in the crude material from the current 
study (table V-2) . Fourfold increases in specific activity were gained 
at each step of fractionation with Sephacryl S-200 and DEAE-cellulose so 
that 44% of the initial lactogenic activity remained with a specific 
activity of 224.2 yg prl-equivalents/mg protein after two steps. Chroma- 
tography with Sephadex G-75 increased purity another 24% so that the 
partially purified preparation of bPL contained 492 yg prl-equivalents 
in 1.77 mg of total protein for a specific activity of 277.8 yg/mg, and 
a biological potency of 5.03 lU/mg based on radioreceptor assay (table 
V-2). In contrast, Bolander and Fellows (1976) began with 19 g of pro- 
tein and isolated 82 yg of total lactogenic activity which had a 
biological potency of only .04 lU/mg (or 1/100 as pure as the prepara- 
tion from the current study). Beckers et al. (1980) isolated 1250 yg 
of lactogenic activity from an initial 42.5 g of crude protein, and their 
preparation had a biological potency of 16.1 lU/mg, or threefold more 
active than the preparation in the current investigation. However, 
their procedure did not provide any information about the physical 
properties of the hormone, such as molecular weight, or isoelectric 
point (Beckers et al. , 1980). This is important to evaluate their 



-179- 



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-180- 



preparation since Lewis et al. (1980) have indicated that exposure of 
growth hormone to several different proteolytic enzymes formed cleavage 
products with greater activity than the native hormone. 

Histological photomicrographs of mammary tissue from the bioassay 
performed in the current investigation demonstrated lactogenic activity 
in the G-75 active fractions (figure V-6) , but only at the highest dose 
utilized (5000 ng prl-equivalents/ml medium). Plate A (figure V-6) 
depicts tissue fixed at the time the rabbit was killed (day 19 of preg- 
nancy and day of culture) and indicated that differentiation of the 
mammary gland had already begun as several alveoli had enlarged lumina 
containing numerous white lipid droplets (figure V-6, A). This was not 
desirable because the change in histology after culture with lactogenic 
hormones was more subtle than if compared to completely undifferentiated 
tissue. However, the majority of the alveoli in the lobules were not 
yet active. Mammary tissue cultured for 48 hr in mediijm containing 
insulin and Cortisol (figure V-6,B) contained only a few alveoli with 
lipid droplets clearly visible, and no lumina looked packed with secre- 
tion. This can be sharply contrasted to tissue cultured in IC and 1000 
ng prolactin/ml (figure V-6,C), Almost all alveoli had lumina in which 
lipid was visible and the majority of lumina were quite distended 
(figure V-6,C), demonstrating a very characteristic "lactogenic response." 
Tissue cultured in medium containing IC and 5000 ng bPL (prl-equivalents) / 
ml also demonstrated a lactogenic response (figure V-6,D) as numerous 
alveoli were noticeably larger than any from tissue cultured in IC 
(figure V-6,B). Secretions within the alveoli of one lobule in figure 
V-6,D all had the eosinophilic staining characteristic of colostrimi, 
while alveoli in other lobules were more heterogeneous with respect to 
the type of secretion (figure V-6,D). 



Figure V-6. Histological photomicrographs of mammary tissue explatits 
prior to the culture period (A) , after 48 hr culture in 
medium containing insulin and Cortisol (B) , after 48 hr of 
culture in insulin, Cortisol, and prolactin (1000 ng/ml) 
(C) , and after 48 hr of culture in medium containing insulin, 
Cortisol, and bovine placental lactogen (5000 ng/ml) (D) . 
Magnification XIOO. 



-182- 




«r^ p«.s^<o ^ *»r '^* 't' 







-183- 



A biochemical response to a stimulus is a better indication of 
lactogenesis than secretory response ratings from the standpoint of 
degree of objectivity. Therefore, rates of mammary tissue fatty acid 
synthesis from 2- C-acetate were determined for tissue -from each of 
the 14 culture media, as well as for tissue from day (which was not 
cultured prior to short term incubation) . Mammary tissue from day 
incorporated .85 nmoles acetate per mg tissue per 2.5 hr (table V-3) , 
and after 48 hr of culture in IC media tissue incorporated only .20 
nmole/mg/2.5 hr. This and histology (figure V-6,A) suggested that mam- 
mary tissue was under some lactogenic stimulus in vivo . Biosynthetic 
response to the media containing IC Prl was curvilinear, as indicated 
by a significant second order least squares regression equation. Tissue 
from cultures with 1 or 10 ng Prl/ml incorporated acetate at virtually 
the same rate as tissue from IC. This was not surprising since 10 ng 
prl/ml would probably not be lactogenic in vivo . However, 100 ng prl/ml 
was definitely lactogenic. At 250 ng prl/ml, tissue incorporated 19.18 
nmoles acetate/mg tissue/2.5 hr Incubation (table V-3). At 500 and 
1000 ng prl/ml, which were pharmacological doses, tissue did not in- 
corporate acetate as rapidly as that incubated in 100 or 250 ng prl, but 
a positive lactogenic response still was evident. In contrast to the 
response to prolactin, tissue in cultures with 1 to 1000 ng bPL/ml did 
not incorporate acetate at a greater rate than tissue cultured in IC 
(table V-3) . Explants from tissue cultured with 5000 ng bPL/ml, however, 
incorporated 4.21 nmoles acetate per mg tissue per 2.5 hr incubation 
(table V-3) . This verified the radioreceptor assay and histological 
response which suggested that bPL was lactogenic. Analysis of variance 
of the biosynthesis data (table V-4) indicated that the medium (IC, 



■184- 



Table V-3. LEAST SQUARES MEANS OF RATES OF ACETATE INCORPORATED INTO 
FATTY ACIDS BY MAMMARY TISSUE EXPLANTS IN SHORT-TERM 
INCUBATION AFTER OR 48 HR OF CULTURE IN MEDIUM CONTAIN- 
ING IC, ICPrl, OR ICbPL^ 



Length of 
Culture (hr) 



Culture 
Mediimi 



Dose of Prl or 
bPL (ng/ml) 



Rate of Acetate 
Incorporation^ 



.85 ± 1.50 



48 



IC 







,20 ± 2.12 



48 
48 
48 
48 
48 
48 



ICPrl 
ICPrl 
ICPrl 
ICPrl 
ICPrl 
ICPrl 



1 

10 

100 

250 

500 

1000 



.28 ± 2.12 

.27 ± 2.12 

14.50 ± 2.12 

19.18 ± 2.12 

9.80 ± 2.12 

11.03 ± 2.12 



48 
48 
48 
48 
48 
48 
48 



ICbPL 
ICbPL 
ICbPL 
ICbPL 
ICbPL 
ICbPL 
ICbPL 



1 

10 

100 

250 

500 

1000 

5000 



.08 ± 2.12 
.11 ± 2.12 
.13 ± 2.12 
.21 ± 2.12 
.19 ± 2.12 
. 18 ± 2.12 
4.21 ± 2.12 



I = Insulin (1 yg/ml) , C = Cortisol (1 pg/ml) , Prl = prolactin (NIAMDD- 
oPRL-14) , and bPL = bovine placental lactogen. 

All values represent mean ± standard error of the mean in nmoles 
2-l'+C-acetate incorporated into fatty acids per mg tissue per 2.5 hr 
incubation. 



-185- 



Table V-4. LEAST SQUARES ANALYSIS OF VARIANCE OF RATES OF ACETATE 

INCORPORATION INTO FATTY ACIDS BY MAMMARY TISSUE EXPLANTS 
IN SHORT-TERM INCUBATION AFTER OR 48 HR OF CULTURE IN 
MEDIUM CONTAINING IC, ICPrl, OR ICbPL^ 



Source 



Medium 

Dose (of Prl or bPL) 

Medium x Dose 

Residual 



df 



2 

6 

5 

33 



MS 



367,0*** 
80.5*'^* 
86.4*** 
13.5 



^I = Insulin, C = Cortisol, Prl = prolactin (NIAMDD-oPRL-14) , and bPL 
bovine placental lactogen. 

b. 



MS = Mean square. 
***P < .001 



■186- 



ICPrl, or ICbPL) and the dose of lactogenic hormone had a significant 
effect on the lipogenic response. There was also a medixim by dose inter- 
action (P < .001, table V-4) . This interaction could best be described 
by second order regression equations which were determined from an 
analysis with media in the model as well as dose (as a continuous vari- 
able) . Y = -14.2323 + 13.5156X - 1.5755X^ where Y is the nmoles acetate 
incorporated per mg per 2.5 hr incubation, and X is the ng prl/ml. For 

bPL the equation Y = 1.7443 - 1.4258X + .2345X^ where X is the ng bPL/ml 

2 
and Y is the lipogenic response. The R for the overall analysis was 

.67 (P < .0001). 

The activity of the G-75 pooled fraction was 5.03 lU/mg according 
to the radioreceptor assay. However, the response to 5000 ng bPL (in 
prl-equivalents/ml) was only 29% of the response to 100 ng prl/ml in 
terms of lipogenesis (table V-3) . There was concern that lyophilization 
of the bPL preparation prior to addition to the culture media had caused 
structural alterations, with a consequent reduction in biological potency. 
Subsequent radioreceptor assay of the culture media confirmed this. No 
lactogenic activity could be determined in media with 1 to 1000 ng bPL 
added, and medium with 5000 ng bPL/ml assayed at 230 ng/ml (or 4.6%). 
Thus the radioreceptor assay agreed with the observation in culture 
that only the 5000 ng bPL/ml medium contained lactogenic activity. 
These results indicated that the bPL preparation was lactogenic (by 
displacement of prolactin from microsomal membranes, histology, and 
fatty acid synthesis) , but that lyophilization induced some change that 
resulted in reduced biological activity. It seems likely that lyophiliza- 
tion caused aggregation or polymer formation since this phenomenon was 
observed in the lyophilized protein used for 2-D PAGE, and since 



-187- 



dimerization had been reported to occur with hPL (Schneider et al. , 
1977). 

In addition to loss of activity by lyophilization there also could 
be a contaminating protease present in the bPL preparation. Because of 
the success of Beckers et al. (1980) in removing contaminating proteins 
by Blue-Sepharose CL-6B resin in their purification of bPL, an additional 
batch of protein was processed. The hormone was purified as before, 
except that after G-75 gel filtration, was applied to a Cibicron-Blue 
column (1.5 x 5 cm) and a 150 ml gradient of to .3 M KCl was run. In 
contrast to the results of Beckers et al. (1980), the bPL preparation 
had no affinity for the resin and eluted in the wash. Of the 148 pg 
prl-equivalents put onto the column only 72 \ig were measured the follow- 
ing day. In an additional 3 days only 21 jag were measured, and 4 days 
later only 4.8 pg of lactogenic activity was detected by radioreceptor 
assay, suggesting that the hormone was very labile, or possibly that a 
protease inhibitor (but not the protease) had been removed. 



D. Summary 

A different approach to the isolation of bPL was attempted in which 

explants of bovine cotyledons were cultured in Minimal Essential Medium 

3 
supplemented with H-leucine to have a marker of the proteins synthe- 
sized in vitro . The advantage of this technique over those previously 
reported was that the initial material was at least 15-fold more active 
than material from cotyledon homogenates. The standardized protocol 
used to purify the hormone included: lyophilization, gel filtration on 
Sephacryl S-200, anion exchange on DEAE-cellulose, lyophilization, and 



-188- 



and gel filtration with Sephzdex G-75. Lactogenic activity was moni- 
tored by radioreceptor assay and a mammary tissue explant culture was 
utilized to test the prolactin-like activity of the partially purified 
hormone. In addition 2-D PAGE was utilized to examine the character of 
the proteins present at various stages of the purification. 

Radioreceptor assay of aliquots of fresh medium indicated that 
cotyledons could synthesize up to 54 yg bPL/ml during a 36 hr incuba- 
tion, suggesting that cotyledon culture was a feasible approach to bPL 
purification. This was especially important because of the many dis- 
crepancies in the literature concerning the physical characteristics of 
bPL, and the large range of concentrations measured in maternal blood. 

Another advantage of the culture system was that proteins synthe- 
sized in vitro could be radiolabeled and detected by f luorography of the 

3 
2-D PAGE gel. In this study the large incorporation of H- leucine into 

non-bPL proteins precluded the development of f luorographs. However, 
it was evident , as purification progressed, that the bPL was radiolabeled 
as elution patterns of radioactivity and lactogenic activity became more 
similar. 

Other than the explant culture technique, procedures used to iso- 
late bPL were conventional, and apparently effective. Appearance of 
proteins with common isoelectric points on 2-D PAGE of the partially 
purified bPL indicated that only slightly acidic proteins remained. 
Appearance of groups of proteins in the gel with similar molecular 
weights but slightly different isoelectric points was not surprising in 
light of the similar nature of human growth hormone (Lewis et al. , 1980) 
and human PL (Chatterjee et al. , 1977). However, the large molecular 
weights of some proteins present in the gel was of great interest. This 



-189- 



provided some insight into the loss of biological activity since the bPL 
preparation was lyophilized prior to 2~D PAGE and prior to bioassay. 
The most densely staining protein on the gel was believed to be bPL 
since the molecular weight (36,000) was similar to that observed for 
the native molecule with Sephadex G-75. The appearance of proteins 
smaller than this probably represented post-translational processing or 
the products of hydrolytic enzyme action on bPL. Proteins larger than 
bPL in the gel may have represented aggregates, or contaminants of some 
kind, or possibly disulf ide-linked dimers and trimers, etc., of the 
monomeric form of bPL. A precedent is the stable dimeric "big placental 
lactogen" previously reported (Schneider et al. , 1977), which maintains 
immunologic activity of the two separate monomers, but assumes the 
activity of a single monomer in radioreceptor assay. If bovine placental 
lactogen forms similar polymeric associations then the activity of the 
hormone would be greatly reduced in radioreceptor assay, and probably 
biological potency in explant culture. It may be prudent to beware of 
such polymeric associations. 

Degree of purification of bPL in the present study (table V-2) 
was comparable to those of other studies. It is significant that for 
the first time, two investigators report a similar molecular weight 
estimate. The 36,000 dalton estimate by SDS electrophoresis and 34,000 
dalton estimate by gel filtration in the current study agreed to that 
reported (32,000 daltons) by Bremel et al. (1979). 

Activity of the partially purified bPL in mammary gland explant 
culture provided further evidence of lactogenic activity, but the true 
biologic potency of the G-75 pooled fractions was not expressed because 
of loss of activity (probably during lyophilization prior to media pre- 
paration) . 



-190- 



Because of the proposed function of bPL to regulate maternal mammary 
development (and possibly fetal development) interest will continue. 
Before pertinent physiological questions can be answered a homogeneous, 
stable preparation of bPL must be developed. In the current study, 
lyophilization did not appear to be very detrimental to bPL until purity 
became greater than 20%. In the future, alternate procedures (such as 
gel filtration for desalting, and concentrating with filtration mem- 
branes as opposed to extended dialysis and lyophilization) are recommended 
to avoid steps which might reduce biological activity. Purified bPL may 
require special storage techniques (such as ultra low temperatures and /or 
favorable reducing conditions). In addition, it would be revealing to 
compare the behavior of bPL subjected to SDS-PAGE and gel filtration 
with, and without, g mercaptoethanol or glycerol to determine the nature 
of the molecule. This would provide information about the tendency of 
bPL to form disulfide or hydrophobic bonds, and therefore suggest 
effective storage and/or handling procedures. 



CHAPTER IV 
GENERAL SUMMARY 

A series of experiments were conducted to examine hormone produc- 
tion by the conceptus or maternal hormone concentrations which reflect 
conceptus production, and the effect of conceptuses on ammary development. 
Two species were compared in these studies: the pig for which there was 
no evidence of a placental lactogen, and the cow for which there was. 

Since few studies had examined mammary development in the pig, the 
initial study of this dissertation was to characterize mammogenesis and 
lactogenesis in gilts. Histology and nucleic acid concentrations of 
gilt mammary tissue indicated that rapid lobulo-alveolar development 
was initiated at midpregnancy and was complete by day 90. There was 
no further increase in DNA concentration between day 90 of pregnancy 
and the fourth day of lactation. From examination of whole mounts, 
Turner (1952) had indicated that growth of the porcine mammary gland 
appeared complete by day 75 of pregnancy. Hacker (1970) did not detect 
a difference in total DNA content of gilt mammary glands from day 110 
of pregnancy through the second day of lactation, so the great majority 
of mammary gland growth (in terms of cell numbers) was complete by day 
90 of pregnancy in gilts. 

Lactogenesis in the pig is similar to that in the rabbit and cow 
in that it occurred in two stages. Stage I lactogenesis occurs between 
days 90 and 105 of pregnancy and is noted by histological appearance of 



-191- 



■192- 



secretion, the beginning of ultrastructural differentiation of mammary 
epithelial cells, and an increased concentration of RNA in mammary tis- 
sue. Stage II lactogenesis occurs in gilts between day 112 of pregnancy 
and the fourth day of lactation as indicated by a further increase in 
RNA, the completion of ultrastructural differentiation as indicated by 
organelles which appeared active in milk component synthesis, and large 
increases in rates of substrate oxidation and incorporation into lipid 
by mammary tissue slices in vitro . 

Results from previous studies and from data in Chapter IV suggest 
that mammogenesis in the pig may be regulated by estrogen produced by 
the conceptus. Estrogen, progesterone, and prolactin act synergistically 
to stimulate mammogenesis in vitro , and estrogen has been shown to induce 
the prolactin receptor in mammary tissue of virgin rats. Progesterone 
and prolactin concentrations during pregnancy were not affected by con- 
ceptuses (Chapter IV), but estrogen concentrations were. In addition, 
increased concentrations of estrogen in maternal plasma were observed 
when the large increase in mammary cell numbers occurred (days 70-90) . 
Therefore, it is possible that estrogen production (or an increasing 
estrogen to progesterone ratio) induced the prolactin receptor in mammary 
tissue which then stimulated proliferation. It also was possible that 
estrogen stimulated mammogenesis by acting directly on the mammary 
epitheliiim through a mechanism which was independent of the prolactin 
receptor. Neither can one rule out the possibility that estrogen in- 
duced some unidentified growth factor which acted on the mammary gland. 
However, the positive correlations between conceptus presence and estro- 
gen production, and increasing estrogen and mammary gland DNA from 
days 70 to 90 suggested that estrogens were involved. 



-193- 



Control of lactogenesis in the pig also is largely unknown. Stage 
I lactogenesis occurred during the period when concentrations of estro- 
gen, and the estrogen to progesterone ratio, were increasing, but before 
"concentrations of prolactin increased. Again, estrogen may act inde- 
pendently to initiate secretion, or estrogen may induce the prolactin 
receptor so that the concentration of prolactin already present could 
initiate secretion. However, progesterone concentrations still were 
elevated at this time, and progesterone is known to be inhibitory to 
lactogenesis. Thus, copious milk secretion did not ensue. After day 
112 of pregnancy, prolactin (Dusza and Kryzmowska, 1981) and glucocorti- 
coid (Killian et al. , 1973) concentrations begin to increase, and pro- 
gesterone concentrations rapidly decline. This changing hormonal milieu 
is associated with stage II lactogenesis and the pronounced increase in 
metabolic activity noted in the current experiment. Based on effects 
demonstrated in mammary tissue culture studies, it is possible that in- 
creasing concentrations of estrogen, prolactin, and glucocorticoids, 
and decreasing concentrations of progesterone, all are involved in the 

regulation of lactogenesis stage II in the pig. In rats and women, ; 

i 

lactogenesis is inhibited effectively until parturition, and is asso- i 

f 
ciated with a decrease in progesterone and placental lactogen concentra- l 

I 

tions. Placental lactogen is lactogenic in vitro (Topper and Freeman, I 

i 

1980) but its effect on lactogenesis in vivo has not been established. I 

The Initial study of this dissertation also established that there was 
a temporary increase in substrate oxidation by mammary tissue at mid- 
pregnancy which declined until the process of lactogenesis began. This 
was believed to be related to the different populations of cells which 
were present in mid versus late gestation, and the need to mobilize the 



-194- 



large store of triglyceride present within the adipocytes. This occurred 
during a period when concentrations of progesterone were high and just 
prior to the increase in plasma concentration of estrogen which con- 
tinues until late gestation. A preferential utilization of acetate (as 
opposed to glucose) by mammary tissue also was noted during this period. 
This mammary adaptation apparently reflects the diabetogenic effect of 
pregnancy observed in pigs by George et al. (1978). Steroid hormones 
may regulate this phenomenon in the pig mammary gland since hypertri- 
glyceridemia and altered glucose metabolism have been observed after 
administration of steroid hormones to human subjects (Spellacy et al. , 
1978). 

Results from Chapter IV of this dissertation showed a significant 
positive relationship between conceptus numbers and maternal estrogen 
concentrations during pregnancy, but not progesterone or prolactin. 
This suggested that there was little, if any, placental production of 
progesterone or prolactin. Correlations among hormone concentrations 
showed that E-SO,, E , and E„ were associated positively with each other, 
but not with prolactin or progesterone. This suggested that estrogen 
probably did not modulate prolactin secretion in the pregnant gilt. 
Total mammary gland DNA was four to five times as great in pregnant gilts 
as it was in pseudopregnant animals indicating that conceptus presence 
was required for normal mammary development. However, there appeared to 
be no additional benefit of having greater than four to seven conceptuses. 
The relationship between conceptus number and total mammary gland DNA 
in gilts was best described by a quadratic equation. However, if the 
pseudopregnant gilts were omitted from the analysis then the quadratic 
relationship was no longer significant. Among pregnant gilts the 



-195- 



relationship between conceptus numbers and total mammary DNA was best 
described by a cubic equation which was of questionable biological sig- 
nificance since only eight gilts were used. Since data were not gathered 
in gilts with less than four but more than one conceptus we do not know 
how many conceptuses are required for full mammogenic response. Con- 
ceptus stimulation of mammary development in the pig (being curvilinear) 
was slightly different from that in the mouse in which DNA content of 
the mammae was linearly related to conceptus numbers (Nagasawa and Yanai, 
1971). In contrast to the mouse or cow, there is no evidence for porcine 
placental lactogen. The curvilinear effect of conceptus number on mam- 
mary development in the pig may support the hypothesis that estrogen of 
conceptus origin stimulates maramogenesis via induction of the prolactin 
receptor in mammary tissue since gilts with eight to 11 conceptuses had 
greater estrogen concentrations than gilts with four to seven conceptuses. 
On the other hand, if may be that the amount of estrogen produced by 
four to seven conceptuses was sufficient to stimulate epithelial cell 
proliferation independently of prolactin, or the quantity needed to 
stimulate some other unidentified growth factor. Evidence to support 
the hypothesis that estrogen stimulates mammogenesis via the prolactin 
receptor was provided by within group correlations. Correlations between 
prolactin and mammary gland wet weights were large and positive in 
groups 2 and 3 which had high concentrations of estrogen. In contrast 
the correlation between prolactin concentration and mammary wet weight 
in group 1 gilts with low concentrations of estrogen was zero. Since 
one of the well documented effects of prolactin is to induce lactogenesls 
and therefore fluid accumulation within the gland, this may suggest that 
the prolactin receptor was very low in tissue of group one gilts. 



-196- 



However, since fluid is only one of the components affecting mammary 
gland wet weight, this cannot be stated with any certainty. 

An additional experiment was performed to examine one aspect of 
the fetal involvement in lactogenesis . Nara and First (1981a, b) recently 
reported that PGF a was involved directly in glucocorticoid-lnduced 
parturition. Therefore the question was posed as to whether lacto- 
genesis could be induced in pseudopregnant gilts by injections of PGF^a 
in the absence of conceptuses. This experiment was of a preliminary 
nature to test usefulness of the pseudopregnant animal as a model for 
study of lactogenesis in the gilt. Results indicated that PGF a induced 
some of the changes indicative of lactogenesis. However, due to numerous 
possible hormonal changes following PGF a injection, i.e., prolactin, 
growth hormone, glucocorticoids, progesterone, and relaxin it was impos- 
sible to delineate the direct role of PGF a in the lactogenic response. 
In addition, reduced mammary growth in pseudopregnant animals lowered 
metabolic responses to treatment since fewer cells were present than 
found in mammary tissue of pregnant animals. Thus, the pseudopregnant 
gilt makes an excellent model to study mammary growth, but numerous dif- 
ficulties make it a poor model to study hormonal control of lactogenesis. 
In vitro culture of mammary tissue seems a more appropriate approach to 
delineate hormonal (and prostaglandin) control of lactogenesis. 

Prior to these studies, there were no reports of a relationship 
between conceptus numbers and mammary development in the pig. This was 
not the case with the cow. In 1976, Bolander and Fellows reported that 
bPL, a hormone with prolactin-like activity, had been purified from 
bovine placentae. Bolander et al. (1976) further reported that concen- 
trations of bPL in maternal serum during gestation were related to 



-197- 



subsequent milk yields in both dairy and beef cows. However, reports 
since that time have indicated that bPL concentrations in maternal circu- 
lation, as measured by radioreceptor or bioassay, were less than 100 ng/ml. 
This contrasted the 1100 ng/ml reported by Bolander et al. (1976) and 
the report that none was present in maternal plasma (Blank et al. , 1977; 
Schellenberg and Friesen, 1981). In addition, four subsequent reports 
of the purification of bPL described preparations with a wide variety of 
molecular weights and biological potencies (table V-1) . A logical 
question to pose was whether bPL really existed. Thatcher et al. (1980) 
and Erb et al. (1980) reported that dairy calf birth weights were asso- 
ciated positively with subsequent milk yields of their dams. There was 
a clear need to try to purify bPL, since this would be the first step 
to determine if this was a mechanism whereby the bovine fetus could 
stimulate mammary gland development, and therefore impart a greater 
milk yield potential upon the dam. 

To enhance the purification of bPL, explants of bovine cotyledons 

from cows in late gestation were incubated in a tissue culture medium 

3 
containing H-leucine so that porteins synthesized in vitro would be 

radiolabeled. Tissue was centrifuged away from the supernatant at the 

end of the culture, and only the supernatant which was enriched in 

secretory proteins was utilized. This greatly reduced contamination 

from structural proteins and possibly from potentially harmful lysosomal 

enzymes. Purification was accomplished by a series of gel filtration 

and anion exchange chromatography columns, and lactogenic activity was 

monitored with a lactogenic radioreceptor assay since prolactln-like 

activity was the function of interest. Results suggested that cotyle- 

donary culture was a feasible approach to the production of bPL. 



-198- 



Approximately 800 mg of cotyledonary explants synthesized up to 795 yg 
of prolactin equivalents after 36 hr of culture; and the starting 
material for purification was 15 to over 100-fold more active than pre- 
vious bPL preparations. After three purification steps, a partially 
purified protein remained with a specific activity of 277.8 ]ig prolactin- 
equivalents per mg protein, or 5.03 lU/mg protein based on radioreceptor 
assay. On 2-D PAGE this preparation appeared as a series of groups of 
proteins with common isoelectric points (pi = 6.2-6.4), but a wide range 
in molecular weights (29,000-100,000 daltons) . It was possible that the 
closely migrating groups of proteins at the same molecular weight but 
slightly different pi were genetic variants since this had been observed 
with growth hormone (Lewis et al., 1980) and human placental lactogen 
(Chatterjee et al., 1977). The most densely staining group of proteins 
were at a molecular weight of 36,000 and were presumed to be bPL since 
a similar molecular weight was observed for the native macromolecule 
upon gel filtration. The reason for proteins with different molecular 
weights was unclear. It was possible that proteins smaller than 36,000 
were fragments of bPL after post-translational processing or hydrolytic 
enzyme cleavage. The larger molecular weight proteins were presumed to 
be aggregates or polymers of bPL since the larger proteins were at the 
same pi as bPL and were too large to have eluted in the fractions pooled 
from Sephadex G-75. In addition, human placental lactogen has a tendency 
to form a dimer via disulfide linkage so there was a precedent for another 
lactogenic hormone of placental origin to do this. An explant culture 
bioassay of semipurified bPL was performed, and the protein like that 
utilized for 2-D PAGE was lyophilized prior to explant culture. While 
lactogenic activity was exhibited, it was reduced greatly compared to 



-199- 



that expected based on radioreceptor activity prior to lyophilization. 
Subsequent radioreceptor assay of the explant culture media indicated 
that indeed some conformational change had occurred which reduced 
biological potency and apparently reflected what was observed in the 
2-D PAGE system. 

Results suggest that bPL has been purified partially and has a 
molecular weight similar to that reported previously by Bremel et al. 
(1974). However, information needs to be acquired about the hormone's 
molecular conformation, such as its tendency to form hydrogen bonds, 
hydrophobic bonds, or disulfide bonds, and how each of these affects 
the biological activity of the hormone. This may provide information 
useful for storage and handling procedures so that maximum biological 
potency can be maintained. 

Numerous questions about bPL remain. From the diversity of species 
which exhibit placental lactogens, it does not appear to be peculiar to 
any particular placental type. Numerous examples of animals with epi- 
theliochorial and hemochorial placentae have been noted (Talamantes, 
1980). It is possible that placental lactogen does not exist in species 
with endothelial chorial placentae but this has not, as yet, been 
thoroughly investigated. 

Concentrations of bPL in maternal plasma of the cow during gesta- 
tion apparently are much lower than originally reported. It could be 
that bPL never reaches the maternal circulation, but is produced to act 
locally to regulate fetal growth or conceptus steroidogenesis. Placental 
lactogens in other species have been shown to play a role in nitrogen 
and potassium retention (Kaplan and Griimbach, 1974), and active trans- 
port across placental membranes (Baser et al. , 1981) . In this manner. 



-200- 



the conceptus could produce a substance which could act locally as a 
growth promoter, or in osmoregulation. The specific site of action is 
certainly a matter of speculation but it would seem logical that the 
substance would bind to the maternal epithelium at the caruncle since 
this is the microvillous area in ruminant placentae. On the other hand, 
one must not rule out the possibility that placental lactogen is pro- 
duced to move in the fetal direction. Since the allantois is a reservoir 
or proteins, sugars, and electrolytes it plays a prominent role in fetal 
nutrition. Furthermore, rapid allantoic fluid volume expansion in early 
pregnancy may require the presence of placental lactogen. Bazer et al. 
(1981) have shown that ergocryptine administration to gilts inhibits 
water accumulation in the porcine allantois early in pregnancy, and 
placental lactogens can bind to the prolactin receptor (Shiu et al. , 
1973) . This may be the primary function of placental lactogen in the 
cow. However, until a purified preparation is obtained, stabilized, 
and used to examine binding and other physiological processes at these 
particular tissues, this remains a matter of speculation. 

Finally, the question still remains as to whether bPL participates 
in mammary development in the cow. Hopefully, a specific radioimmuno- 
assay will be available in the near future to more accurately quantify 
concentrations of bPL in maternal plasma. If bPL cannot be measured, 
then a likely candidate for further investigation would be estrogens 
since they are produced by the bovine conceptus, as well. 



APPENDIX I 
AMONG ANIMAL CORRELATION BETWEEN MEAN HORMONE CONCENTRATION 



Estrogen- 
Sulfate 


Estrone 


Estradiol 


Progesterone 


Prolactin 


Estrogen- 
Sulfate 


, 75*** 


.59* 


-.07 


-.14 


Estrone 




.67** 


.07 


-.20 


Estradiol 






-.08 


-.43 


Progesterone 








0.0 



*P < .05; **P < .01; ***P < .001 



-201- 



APPENDIX II 

AMONG ANIMAL CORRELATIONS BETWEEN MEAN HORMONE CONCENTRATIONS 
(DAY 10-90) AND MAMMARY VARIABLES IN GILTS WITH 0, 4-7, 

OR 8-11 FETUSES 





Estrogen- 










Response 


Sulfate 


Estrone 


Estradiol 


Progesterone 


Prolactin 


Mammary wet weight 


.668** 


.078 


-.107 


.097 


.481"^ 


DNA (pg/g DFFT) 


.485^ 


.504"^ 


.669** 


.032 


-.263 


Total DNA 


.713** 


.189 


.204 


.024 


.317 


RNA (yg/g DFFT) 


.583* 


.363 


.310 


-.053 


-.070 


Total RNA 


.690** 


.106 


,032 


-.005 


.386 


RNA:DNA ratio 


.525^ 


.100 


-.236 


-.222 


.181 



P < .10; *P < .05; **P < .01 



-202- 



APPENDIX III 

AMONG ANIMAL CORRELATIONS BETWEEN MEAN HORMONE CONCENTRATIONS 
(DAY 70-90) AND MAMMARY VARIABLES IN GILTS WITH 0, 4-7, 

OR 8-11 FETUSES 





Estrogen- 










Response 


Sulfate 


Estrone 


Estradiol 


Progesterone 


Prolactin 


Mammary wet weight 


.565* 


.133 


-.028 


-.015 


.063 


DNA (yg/g DFFT) 


.469"^ 


.352 


.684** 


-.254 


-.236 


Total DNA 


.619* 


.178 


.251 


-.132 


-.091 


RNA (ug/g DFFT) 


.563* 


.364 


.372 


-.355 


-.099 


Total RNA 


.602* 


.152 


.092 


-.157 


-.022 


RNA: DNA ratio 


.516^ 


.257 


-.115 


-.419 


.081 



P < .10; *P < .05; **P < .01 



-203- 



APPENDIX IV 

WITHIN-GROUP CORRELATIONS BETWEEN MEAN HORMONE CONCENTRATIONS 

AND MAMMARY VARIABLES IN GILTS WITH 0, 4-7, 

OR 8-11 FETUSES 



Estrogen- 
Sulfate Estrone Estradiol Progesterone Prolactin 



Group 1 



Mammary wet weight 

DNA (pg/g DFFT) 

Total DNA 

RNA (pg/g DFFT) 

Total RNA 

RNA: DNA ratio 



Group 2 

Mammary wet weight 

DNA (yg/g DFFT) 

Total DNA 

RNA (pg/g DFFT) 

Total RNA 

RNA: DNA ratio 



Group 3 

t 

Mammary wet weight -.275 -.929, 

DNA (yg/g DFFT) .565 .936 

Total DNA -.036 -.881 

RNA (ug/g DFFT) .837 .624 

Total RNA .028 -.839 

RNA:DNA ratio .168 -.256 



.649 


-.178 


.288 


-.355 


.009 


.640 ■ 


.270 


.500 


-.061 


.380 


.682 


-.092 


.277 


-.411 


.103 


.671 


.035 


.356 


-.351 


.227 


.675 


-.139 


.232 


-.472 


.068 


.682 


-.186 


.071 


-.657 


.039 



-.654 


-.789 


-.799 


.242 


.914 


.950* 


.309 


.814 


-.376 


-.882 


.201 


-.749 


-.099 


-.379 


.476 


.708 


-.121 


.364 


.111 


-.804 


-.287 


-.881 


-.546 


-.017 


.775 


-.875 


-.666 


-.965* 


.572 


.819 



-.875 


-.489 


.656 


.964 


.106 


-.829 


-.864 


-.693 


.773 


.650 


-.454 


-.466 


-.817 


-.734 


.734 


-.127 


-.494 


-.090 



'P < .10; *P < .05 
^ = 5. 
^N = 4. 



-205- 



APPENDIX V 

COMPOSITION OF MINIMUM ESSENTIAL MEDIUM UTILIZED FOR 
COTYLEDON EXPLANT CULTURE 



Component 


mg per liter 


CaCl2-2H20 


256 


KCl 


400 


MgSO^-yH^O 


200 


NaCl 


6800 


NaHCO 


2200 


NaH„PO, -H^O 
2 4 2 


140 


Glucose 


5000 



D-Ca pantothenate 
choline CI 
folic acid 
i- inositol 
nicotinamide 
pyridoxal HCl 
riboflavin 
thiamine HCl 

Component 
penicillin 
streptomycin 
amphotericin B 



1.0 
1.0 
1.0 
1.0 
1.0 
1.0 
0.1 
1.0 

Amount per liter 
100,000 U 
100,000 U 

.25 mg 



Component 

L-arginine HCl 

L-cystine 

L-glutamine 

L-histidine HCl 

L-isoleucine 

L-leucine 

L-lysine HCl 

L-methionine 

L-phenylalanine 

L-threonine 

L-tryptophan 

L-tryosine 

D-valine 

L-alanine 

L-asparagine • HO 

L-aspartic acid 

L-glutamic acid 

glycine 

L-proline 

L- serine 



mg per liter 
126.0 
24.0 
292.0 
42.0 
52.0 
52.0 
73.0 
15.0 
32.0 
48.0 
10.0 
36.0 
92.0 

8.9 
15.0 
13.3 
14.7 

7.5 
11.5 
10.5 



insulin 



200 IT 



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APPENDIX VI 

PROCEDURE FOR PREPARATION OF MICROSOMAL FRACTION FROM 
LACTATING RABBIT MAMMARY GLAND FOR USE IN 
LACTOGENIC RADIORECEPTOR ASSAY 



Buffers ; 



.25 M sucrose, 30 mM tris-HCl, pH 7.5 (for homogenization) and 
25 mM tris-HCl, 10 mM CaCl , pH 7.76 (for storage) 

Tissue : 

1. Lactating rabbits received 1 mg injections subcutaneously of ergo- 
cryptine in 50% ethanol:50% water, 48, 36, 24, and 12 hr prior to 
exsanguination on day 10 of lactation. 

2. After cervical dislocation the mammary tissue was removed leaving 
muscle and adipose tissue behind, and mammary tissue placed in ice- 
cold tris-sucrose buffer. 

3. Tissue was minced with scissors and maintained on ice until homogenized 
in two volumes of tris-sucrose buffer (ice-cold) using 3 x 15 sec 
bursts with the Polytron (Brinkman Instrtiments) on a speed setting 

of 7 or 8. 

4. Homogenized material was filtered through two, then four layers, of 
cheesecloth and distributed into eight centrifuge tubes. 

5. Filtrate was centrifuged at 15,000 g (11,100 rpm with SS-34 head) for 
20 min at 4 C. 

6. The supernatant from step 5 was centrifuged at 100,000 g for 60 min 
at 4 C (31,000 rpm with T-865 head). 

7. The microsomal pellet from step six was resuspended in 25 mM Tris, 
10 mM CaCl2 Buffer using a nylon: glass homogenizer to get pellet 
into solution. 

8. A Lowry assay was run on several dilutions of the resuspended 
material and additional buffer added to freeze the membrane at 
5 mg protein per ml. 

9. The preparation was quickly frozen in 1, 2, and 3 ml volumes. 



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APPENDIX VII 
PROCEDURES AND VALIDATION FOR LACTOGENIC RADIORECEPTOR ASSAY 



Buffer : 

25 mM Tris-HCl, 10 mM CaCl^, .1% (wt/vol) BSA, pH 7.76 at 5 C. 

Membrane preparation was diluted 1:2, homogenized with a glass: glass 
homogenizer, and 100 yl (representing 250 lag protein) of the preparation 
was added to each assay tube (12 x 75 mm), along with 50,000 cpm of 

I-prolactin. All dilutions and additions were made with 25 mM Tris 
buffer. Native unlabeled prolactin was added to yield 10, 25, 50, 100, 
250, and 500 ng per tube; all standard curves were run in duplicate. 
Addition of membrane preparation, labeled and unlabeled prolactin, and 
buffer yielded a final reaction volume of 500 yl. Incubation was carried 
out at 5 C for 24-48 hr, and terminated with the addition of 3 ml of 
ice-cold buffer. Tubes were centrifuged immediately for 30 min at 
1700 g (3000 rpm with the HS-4 head) , supernatant (free hormone) de- 
canted, and tubes inverted for a few minutes. After blotting the tubes 
on absorbent paper the bound hormone remaining in the pellet was counted 
in a gamma counter. 

Recovery of added mass of prolactin was determined by adding 10, 
100, 250, or 500 ng prolactin (in duplicate) to 25 mM Tris buffer (300 
uD before addition of membrane preparation and labeled prolactin (as 
above) . The mass of prolactin measured is indicated below and is de- 
scribed by the equation Y = 19.57 + . 82X where Y = ng prolactin measured, 
and X = ng prolactin added. 



Validation of Recovery of Added Mass 
N ng prolactin added ng prolactin measured % recovery 



2 10 12 120 

2 100 102 102 

2 250 255 102 

2 500 415 83 



Prolactin added was NIH-PB9. 



-211- 



-212- 



Crossreactivity of Various Hormones 
(when added at 10, 100, 1000, and 10,000 ng/tube) 



Hormone Cross Reactivity 



bovine LH 

bovine FSH 

thyroxine 

insulin 

bovine GH 4.3 

human PL 3.2 



^Crossreactivity calculated with 215 ng prolactin as numerator, and the 
amount of the tested hormone required to displace 50% of the total bind- 
ing tube as the denominator. 

hPL was isolated by and was a gift from William Buhi (Obstetrics and 
Gynecology Department of the University of Florida. 



APPENDIX VIII 

PROCEDURE FOR CONSTRUCTING SELECTIVITY CURVES FOR DETERMINATION 
OF MOLECULAR WEIGHTS BY GEL FILTRATION 



A selectivity curve is determined by plotting the log (base 10) 
of the molecular weight versus the corresponding K value for several 
standard proteins of known molecular weight. The K value represents 
the fraction of the stationary gel volume which is available for dif- 
fusion of a given solute species and is determined by: 



Ve - Vo 



"AV Vt - Vo 

where 

Ve is the elution volume of the protein in question, 

Vo is the void volume (elution volume of Blue Dextran) , and 

2 
Vt is the total bed volume determined by irr h. 

Gel filtration calibration kits may be purchased from Pharmacia 
for constructing selectivity curves. 



-213- 



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BIOGRAPHICAL SKETCH 

Ronald Scott Kensinger was born on October 24, 1953, in Elmhurst, 
Illinois. He is the third of three sons raised by Dean G. and Velma F. 
Kensinger. After completing primary school in Westchester, Illinois, 
he attended Proviso West Township High School in Hillside, Illinois, 
graduating in 1971. From there, he attended the University of Illinois 
in Champaign-Urbana, receiving a Bachelor of Science degree in animal 
science in 1975. The decision to obtain a Master of Science degree led 
him to the Department of Dairy Science at the University of Illinois. 
In 1977 he moved to the University of Florida to pursue a Doctor of 
Philosophy degree in animal science. He was married to Margaret McKelvey 
Heekin on January 24, 1981, and the following winter accepted a position 
as Assistant Professor in the Dairy and Animal Science Department at 
The Pennsylvania State University. He is a member of the American Dairy 
Science Association, American Society of Animal Science, the Endocrine 
Society, and Sigma Xi. 



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I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



y 






Robert J. Collier, Chairman 
Associate Professor of Animal Science 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 




Fuller W. Bazer 

Professor of Animal Science 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



William W. Thatcher 
Professor of Dairy Science 



I certify that I have, read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



i / 



H. Herbert Head 

Associate Professor of Dairy Science 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is fully 
adequate, in scope and quality, as a dissertation for the degree of 
Doctor of Philosophy. 



Donald Caton 

Professor of Obstetrics and Gynecology, 

and Anesthesiology 



This dissertation was submitted to the Graduate Faculty of the College 
of Agriculture and to the Graduate Council, and was accepted as partial 
fulfillment of the requirements for the degree of Doctor of Philosophy. 



May 3 1982 






/"..^ 



Dean/;' College of Agrielilture 



Dean for Graduate Studies and Research